615 lines
84 KiB
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615 lines
84 KiB
XML
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<mods:title id="54D7ADC98CFA42536FC3011AE4E9589D">Probing of the plasticity of the active site in pinene synthase elucidates its potential evolutionary mechanism</mods:title>
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<mods:namePart id="32908597D574631B18AB41F6A84732AF">Xu, Jingwei</mods:namePart>
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<mods:namePart id="CBE54B71A55A557234A740C86A540D46">Peng, Guanzu</mods:namePart>
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<mods:namePart id="BE9648789A563146C1716E5554C067CF">Xu, Jinkun</mods:namePart>
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<mods:namePart id="C6DB3CB0920C64BA14C05CA9839F477F">Li, Yi</mods:namePart>
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<mods:namePart id="C5E85DE8AE8975198A31C58797C25117">Tong, Li</mods:namePart>
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<mods:namePart id="AF0EE9C2F45FF8A505E3B191A2ECB99F">Yang, Dong</mods:namePart>
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<mods:title id="54D60934C193A7A831B6D962DBE71E11">Phytochemistry</mods:title>
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<mods:date id="B852446543DFBB5BCA6F7EB66F50B94B">2021</mods:date>
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<treatment id="03AD87AFFFF8FFB5120FFDB23C39FA3A" ID-DOI="http://doi.org/10.5281/zenodo.8301284" ID-Zenodo-Dep="8301284" LSID="urn:lsid:plazi:treatment:03AD87AFFFF8FFB5120FFDB23C39FA3A" httpUri="http://treatment.plazi.org/id/03AD87AFFFF8FFB5120FFDB23C39FA3A" lastPageId="3" lastPageNumber="4" pageId="1" pageNumber="2">
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<subSubSection id="C31E6532FFF8FFB7120FFDB23D34FDD3" box="[818,1326,567,587]" pageId="1" pageNumber="2" type="nomenclature">
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<heading id="D0F381D5FFF8FFB7120FFDB23D34FDD3" bold="true" box="[818,1326,567,587]" fontSize="36" level="1" pageId="1" pageNumber="2" reason="1">
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<emphasis id="B970EAABFFF8FFB7120FFDB23D34FDD3" bold="true" box="[818,1326,567,587]" italics="true" pageId="1" pageNumber="2">
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2.1. Homology modeling of
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<taxonomicName id="4C044D3AFFF8FFB7150EFDB23C86FDD3" ID-CoL="6TKNH" ID-ENA="35924" authority="Pall." box="[1075,1180,567,587]" class="Magnoliopsida" family="Paeoniaceae" genus="Paeonia" kingdom="Plantae" order="Saxifragales" pageId="1" pageNumber="2" phylum="Tracheophyta" rank="species" species="lactiflora">P. lactiflora</taxonomicName>
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pinene synthase
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</emphasis>
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</heading>
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</paragraph>
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<paragraph id="8BBB36B9FFF8FFB4126CFDE5399EFED7" blockId="1.[818,1488,623,754]" lastBlockId="2.[100,770,148,334]" lastPageId="2" lastPageNumber="3" pageId="1" pageNumber="2">
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The crystal structures of pinene synthases have not been reported. Therefore, we constructed a homolog model of
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<taxonomicName id="4C044D3AFFF8FFB715CEFD063D46FD07" box="[1267,1372,651,671]" class="Magnoliopsida" family="Paeoniaceae" genus="Paeonia" kingdom="Plantae" order="Saxifragales" pageId="1" pageNumber="2" phylum="Tracheophyta" rank="species" species="lactiflora">
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<emphasis id="B970EAABFFF8FFB715CEFD063D46FD07" bold="true" box="[1267,1372,651,671]" italics="true" pageId="1" pageNumber="2">P. lactiflora</emphasis>
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</taxonomicName>
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pinene synthase using the crystal structure of S-limonene synthase as the template. In order to investigate the catalytic mechanism, we docked three carbocation intermediates (terpinyl, pinyl and thujyl cations) into its active site (
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<figureCitation id="133F2A3CFFFBFFB411AFFF1E38EBFF3E" box="[146,241,148,167]" captionStart="Fig" captionStartId="1.[100,130,1888,1905]" captionTargetBox="[188,1399,797,1859]" captionTargetId="figure-487@1.[187,1400,795,1860]" captionTargetPageId="1" captionText="Fig. 1. a, The proposed reaction mechanism for the synthesis of limonene, pinene and sabinene. b, The structural model of pinene synthase docked with the terpinyl (left), pinyl (middle) and thujyl (right) cations. Carbocations are colored in magenta (terpinyl), wheat (pinyl) and cyan (thujyl). Other atoms are colored according to their types (red: oxygen; yellow: carbon; blue: nitrogen; orange: phosphorus; green: magnesium). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)" figureDoi="http://doi.org/10.5281/zenodo.8291068" httpUri="https://zenodo.org/record/8291068/files/figure.png" pageId="2" pageNumber="3">Fig. 1B–D</figureCitation>
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). The docking results suggest that carbocations bind to a similar location in the active pocket. Nearby residues include R305, S311, W314, I335, I338, N339, D342, Y417, I443, A444, I448, F482, D486, S491 and H571. Some of these residues could make hydrophobic interactions with the carbon skeleton of carbocations. Certain polar or aromatic residues may stabilize the positive charge and influence the product profile of the enzyme.
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</paragraph>
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<caption id="DF7B6631FFF8FFB71159F8EA3A5DF824" ID-DOI="http://doi.org/10.5281/zenodo.8291068" ID-Zenodo-Dep="8291068" httpUri="https://zenodo.org/record/8291068/files/figure.png" pageId="1" pageNumber="2" startId="1.[100,130,1888,1905]" targetBox="[188,1399,797,1859]" targetPageId="1" targetType="figure">
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<paragraph id="8BBB36B9FFF8FFB71159F8EA3A5DF824" blockId="1.[100,1488,1888,1981]" pageId="1" pageNumber="2">
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<emphasis id="B970EAABFFF8FFB71159F8EA3886F8E8" bold="true" box="[100,156,1888,1905]" pageId="1" pageNumber="2">Fig. 1.</emphasis>
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a, The proposed reaction mechanism for the synthesis of limonene, pinene and sabinene.
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<emphasis id="B970EAABFFF8FFB712A9F8EA3B85F8E9" bold="true" box="[916,927,1888,1904]" pageId="1" pageNumber="2">b</emphasis>
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, The structural model of pinene synthase docked with the terpinyl (left), pinyl (middle) and thujyl (right) cations. Carbocations are colored in magenta (terpinyl), wheat (pinyl) and cyan (thujyl). Other atoms are colored according to their types (red: oxygen; yellow: carbon; blue: nitrogen; orange: phosphorus; green: magnesium). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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</paragraph>
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</caption>
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<paragraph id="8BBB36B9FFFBFFB41159FEF63967FE32" blockId="2.[100,721,380,427]" pageId="2" pageNumber="3">
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<emphasis id="B970EAABFFFBFFB41159FEF63967FE32" bold="true" italics="true" pageId="2" pageNumber="3">2.2. Alanine scanning mutagenesis identifies mutations that cause an increase in sabinene production</emphasis>
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</paragraph>
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<paragraph id="8BBB36B9FFFBFFB411B9FE5B398EFD63" blockId="2.[100,771,464,762]" pageId="2" pageNumber="3">
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We first identified residues within 5
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from the docked carbocations and performed alanine-scanning mutagenesis on them. Most of these mutations do not show dramatic changes in their product profiles. However, two mutations show interesting alteration in the product profile. One of them,
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482
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, essentially converts the enzyme to sabinene synthase. About 90.8% of its products is sabinene, while 97.7% of the products of the WT enzyme is α- pinene (
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<tableCitation id="C6860302FFFBFFB410D3FDFD3A2CFD12" box="[494,566,631,651]" captionStart="Table 1" captionStartId="2.[100,150,1512,1528]" captionTargetPageId="2" captionText="Table 1 Major products (%±SD) of WT α-pinene synthase and enzymes with mutations." httpUri="http://table.plazi.org/id/DF7B6631FFFBFFB41159FA623B1DF98A" pageId="2" pageNumber="3" tableUuid="DF7B6631FFFBFFB41159FA623B1DF98A">Table 1</tableCitation>
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,
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<figureCitation id="133F2A3CFFFBFFB4137EFDFD3AB9FD12" box="[579,675,631,651]" captionStart="Fig" captionStartId="3.[100,130,1203,1220]" captionTargetBox="[188,1399,149,1175]" captionTargetId="figure-521@3.[187,1400,148,1176]" captionTargetPageId="3" captionText="Fig. 2. Converting pinene synthase to sabinene synthase by mutations on 482 position. Chromatograms in a, b, c, d and e show the GC-MS analysis of terpene products for WT, F482A, F482I, F482V and F482T, respectively. The x-axis is the retention time and the y-axis is the relative abundance of each species. The numbers in each peak correspond to α-pinene (1), sabinene (2) and limonene (3)." figureDoi="http://doi.org/10.5281/zenodo.8291070" httpUri="https://zenodo.org/record/8291070/files/figure.png" pageId="2" pageNumber="3">Fig. 2A–B</figureCitation>
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). Another mutation,
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335
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, also causes an increase in sabinene production, though not as dramatic as
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482
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<collectionCode id="ED15AE7CFFFBFFB41002FD253955FD5B" box="[319,335,687,706]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="2" pageNumber="3" type="Herbarium">A</collectionCode>
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(
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<tableCitation id="C6860302FFFBFFB41066FD2539BAFD5B" box="[347,416,687,706]" captionStart="Table 1" captionStartId="2.[100,150,1512,1528]" captionTargetPageId="2" captionText="Table 1 Major products (%±SD) of WT α-pinene synthase and enzymes with mutations." httpUri="http://table.plazi.org/id/DF7B6631FFFBFFB41159FA623B1DF98A" pageId="2" pageNumber="3" tableUuid="DF7B6631FFFBFFB41159FA623B1DF98A">Table 1</tableCitation>
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). Importantly, although both mutants show an altered product profile, their overall activities are only moderately decreased (
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<tableCitation id="C6860302FFFBFFB4107DFD6D399DFD63" box="[320,391,743,762]" captionStart="Table 1" captionStartId="2.[100,150,1512,1528]" captionTargetPageId="2" captionText="Table 1 Major products (%±SD) of WT α-pinene synthase and enzymes with mutations." httpUri="http://table.plazi.org/id/DF7B6631FFFBFFB41159FA623B1DF98A" pageId="2" pageNumber="3" tableUuid="DF7B6631FFFBFFB41159FA623B1DF98A">Table 1</tableCitation>
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).
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</paragraph>
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<paragraph id="8BBB36B9FFFBFFB41159FCA23AF5FCA2" blockId="2.[100,751,808,828]" box="[100,751,808,828]" pageId="2" pageNumber="3">
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<emphasis id="B970EAABFFFBFFB41159FCA23AF5FCA2" bold="true" box="[100,751,808,828]" italics="true" pageId="2" pageNumber="3">2.3. Phenylalanine at the 482 position is required for pinene production</emphasis>
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</paragraph>
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<paragraph id="8BBB36B9FFFBFFB411B9FCEB3AD0FB47" blockId="2.[100,770,864,1469]" pageId="2" pageNumber="3">
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We hypothesized that the aromatic ring of
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482 might play a role in stabilizing the pinyl cation. To investigate this possibility, we mutated
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482 to Val and Ile, two nonpolar residues with different side chain sizes. We also mutated it to Thr, Arg and Trp, representing polar, charged or non-Phe aromatic residues. The results suggest that any mutations that changes the phenylalanine at this position leads to an increase in sabinene production and almost abolishes the α- pinene production (
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<tableCitation id="C6860302FFFBFFB411E6FBA93938FBAE" box="[219,290,1059,1079]" captionStart="Table 1" captionStartId="2.[100,150,1512,1528]" captionTargetPageId="2" captionText="Table 1 Major products (%±SD) of WT α-pinene synthase and enzymes with mutations." httpUri="http://table.plazi.org/id/DF7B6631FFFBFFB41159FA623B1DF98A" pageId="2" pageNumber="3" tableUuid="DF7B6631FFFBFFB41159FA623B1DF98A">Table 1</tableCitation>
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). Replacing phenylalanine with nonpolar residues (
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482
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,
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482V) all effectively convert the enzyme to sabinene synthase (
|
||
<figureCitation id="133F2A3CFFFBFFB41151FBD138D5FBF7" box="[108,207,1115,1135]" captionStart="Fig" captionStartId="3.[100,130,1203,1220]" captionTargetBox="[188,1399,149,1175]" captionTargetId="figure-521@3.[187,1400,148,1176]" captionTargetPageId="3" captionText="Fig. 2. Converting pinene synthase to sabinene synthase by mutations on 482 position. Chromatograms in a, b, c, d and e show the GC-MS analysis of terpene products for WT, F482A, F482I, F482V and F482T, respectively. The x-axis is the retention time and the y-axis is the relative abundance of each species. The numbers in each peak correspond to α-pinene (1), sabinene (2) and limonene (3)." figureDoi="http://doi.org/10.5281/zenodo.8291070" httpUri="https://zenodo.org/record/8291070/files/figure.png" pageId="2" pageNumber="3">Fig. 2C–D</figureCitation>
|
||
). More than 90% of products made by these mutants are sabinene (
|
||
<tableCitation id="C6860302FFFBFFB411FAFBFD3915FB13" box="[199,271,1143,1162]" captionStart="Table 1" captionStartId="2.[100,150,1512,1528]" captionTargetPageId="2" captionText="Table 1 Major products (%±SD) of WT α-pinene synthase and enzymes with mutations." httpUri="http://table.plazi.org/id/DF7B6631FFFBFFB41159FA623B1DF98A" pageId="2" pageNumber="3" tableUuid="DF7B6631FFFBFFB41159FA623B1DF98A">Table 1</tableCitation>
|
||
). Interestingly,
|
||
<collectionCode id="ED15AE7CFFFBFFB41094FBFD39AFFB13" box="[425,437,1143,1162]" country="USA" lsid="urn:lsid:biocol.org:col:15707" name="Field Museum of Natural History, Botany Department" pageId="2" pageNumber="3" type="Herbarium">F</collectionCode>
|
||
482T also predominantly produces sabinene, which seems to suggest that a polar residue here is not sufficient to stabilize the pinyl cation (
|
||
<tableCitation id="C6860302FFFBFFB41088FB2539E7FB5B" box="[437,509,1199,1218]" captionStart="Table 1" captionStartId="2.[100,150,1512,1528]" captionTargetPageId="2" captionText="Table 1 Major products (%±SD) of WT α-pinene synthase and enzymes with mutations." httpUri="http://table.plazi.org/id/DF7B6631FFFBFFB41159FA623B1DF98A" pageId="2" pageNumber="3" tableUuid="DF7B6631FFFBFFB41159FA623B1DF98A">Table 1</tableCitation>
|
||
,
|
||
<figureCitation id="133F2A3CFFFBFFB41331FB253A49FB5B" box="[524,595,1199,1218]" captionStart="Fig" captionStartId="3.[100,130,1203,1220]" captionTargetBox="[188,1399,149,1175]" captionTargetId="figure-521@3.[187,1400,148,1176]" captionTargetPageId="3" captionText="Fig. 2. Converting pinene synthase to sabinene synthase by mutations on 482 position. Chromatograms in a, b, c, d and e show the GC-MS analysis of terpene products for WT, F482A, F482I, F482V and F482T, respectively. The x-axis is the retention time and the y-axis is the relative abundance of each species. The numbers in each peak correspond to α-pinene (1), sabinene (2) and limonene (3)." figureDoi="http://doi.org/10.5281/zenodo.8291070" httpUri="https://zenodo.org/record/8291070/files/figure.png" pageId="2" pageNumber="3">Fig. 2E</figureCitation>
|
||
). Other mutations (
|
||
<collectionCode id="ED15AE7CFFFBFFB41157FB41386DFB47" box="[106,119,1227,1246]" country="USA" lsid="urn:lsid:biocol.org:col:15707" name="Field Museum of Natural History, Botany Department" pageId="2" pageNumber="3" type="Herbarium">F</collectionCode>
|
||
482
|
||
<collectionCode id="ED15AE7CFFFBFFB411A6FB4138B6FB47" box="[155,172,1227,1246]" country="USA" lsid="urn:lsid:biocol.org:col:14792" name="Yale University" pageId="2" pageNumber="3" type="Herbarium">Y</collectionCode>
|
||
,
|
||
<collectionCode id="ED15AE7CFFFBFFB41188FB4138DBFB47" box="[181,193,1227,1246]" country="USA" lsid="urn:lsid:biocol.org:col:15707" name="Field Museum of Natural History, Botany Department" pageId="2" pageNumber="3" type="Herbarium">F</collectionCode>
|
||
482
|
||
<collectionCode id="ED15AE7CFFFBFFB411D9FB4138E9FB47" box="[228,243,1227,1246]" country="Chile" name="Departamento de Geologia, Universidad de Chile" pageId="2" pageNumber="3">R</collectionCode>
|
||
and
|
||
<collectionCode id="ED15AE7CFFFBFFB4101FFB413935FB47" box="[290,303,1227,1246]" country="USA" lsid="urn:lsid:biocol.org:col:15707" name="Field Museum of Natural History, Botany Department" pageId="2" pageNumber="3" type="Herbarium">F</collectionCode>
|
||
482
|
||
<collectionCode id="ED15AE7CFFFBFFB4106CFB41397DFB47" box="[337,359,1227,1246]" country="Austria" lsid="urn:lsid:biocol.org:col:15588" name="Naturhistorisches Museum Wien" pageId="2" pageNumber="3" type="Herbarium">W</collectionCode>
|
||
) retain very little activities (
|
||
<tableCitation id="C6860302FFFBFFB4134BFB413AA7FB47" box="[630,701,1227,1246]" captionStart="Table 1" captionStartId="2.[100,150,1512,1528]" captionTargetPageId="2" captionText="Table 1 Major products (%±SD) of WT α-pinene synthase and enzymes with mutations." httpUri="http://table.plazi.org/id/DF7B6631FFFBFFB41159FA623B1DF98A" pageId="2" pageNumber="3" tableUuid="DF7B6631FFFBFFB41159FA623B1DF98A">Table 1</tableCitation>
|
||
).
|
||
</paragraph>
|
||
<paragraph id="8BBB36B9FFFBFFB411B9FB6D3A81FA1C" blockId="2.[100,770,864,1469]" pageId="2" pageNumber="3">
|
||
In order to verify that the mutant enzymes indeed produce sabinene, we analyzed the reaction product of
|
||
<collectionCode id="ED15AE7CFFFBFFB410F1FA8939C2FA8F" box="[460,472,1283,1302]" country="USA" lsid="urn:lsid:biocol.org:col:15707" name="Field Museum of Natural History, Botany Department" pageId="2" pageNumber="3" type="Herbarium">F</collectionCode>
|
||
482L using the high resolution hybrid quadrupole-orbitrap GC-MS/MS system (
|
||
<figureCitation id="133F2A3CFFFBFFB41362FA943AD6FAAB" box="[607,716,1310,1330]" captionStart="Fig" captionStartId="4.[100,130,1910,1927]" captionTargetBox="[348,1238,149,1881]" captionTargetId="figure-7@4.[348,1239,148,1882]" captionTargetPageId="4" captionText="Fig. 3. Hybrid quadrupole-orbitrap GC-MS/MS. a shows the chromatogram of terpene products of F482L. The elution peak corresponding to sabinene is labeled. The mass spectrum of the product eluted at 9.29 min from a is shown in b. The mass spectrum of the standard sabinene is shown in c." figureDoi="http://doi.org/10.5281/zenodo.8291072" httpUri="https://zenodo.org/record/8291072/files/figure.png" pageId="2" pageNumber="3">Fig. 3A–C</figureCitation>
|
||
). By comparing with the sabinene standard, the peak eluted at 9.29 min was identified as sabinene. Its mass spectrum was measured and could be readily matched with that of sabinene standard (
|
||
<figureCitation id="133F2A3CFFFBFFB4130FFAF83A8AFA1C" box="[562,656,1394,1414]" captionStart="Fig" captionStartId="4.[100,130,1910,1927]" captionTargetBox="[348,1238,149,1881]" captionTargetId="figure-7@4.[348,1239,148,1882]" captionTargetPageId="4" captionText="Fig. 3. Hybrid quadrupole-orbitrap GC-MS/MS. a shows the chromatogram of terpene products of F482L. The elution peak corresponding to sabinene is labeled. The mass spectrum of the product eluted at 9.29 min from a is shown in b. The mass spectrum of the standard sabinene is shown in c." figureDoi="http://doi.org/10.5281/zenodo.8291072" httpUri="https://zenodo.org/record/8291072/files/figure.png" pageId="2" pageNumber="3">Fig. 3B–C</figureCitation>
|
||
).
|
||
</paragraph>
|
||
<paragraph id="8BBB36B9FFFBFFB411B9FA043BDCFEF3" blockId="2.[100,770,864,1469]" lastBlockId="2.[818,1488,148,362]" pageId="2" pageNumber="3">
|
||
It is interesting to note that replacement of
|
||
<collectionCode id="ED15AE7CFFFBFFB4131BFA043A29FA38" box="[550,563,1422,1441]" country="USA" lsid="urn:lsid:biocol.org:col:15707" name="Field Museum of Natural History, Botany Department" pageId="2" pageNumber="3" type="Herbarium">F</collectionCode>
|
||
482 to other aromatic residues (Tyr or Trp) causes a drastic decrease in enzyme activities. To investigate this issue, we first docked the pinyl cation into the active pockets of WT and
|
||
<collectionCode id="ED15AE7CFFFBFFB412DBFF3A3BE9FF5A" box="[998,1011,176,195]" country="USA" lsid="urn:lsid:biocol.org:col:15707" name="Field Museum of Natural History, Botany Department" pageId="2" pageNumber="3" type="Herbarium">F</collectionCode>
|
||
482
|
||
<collectionCode id="ED15AE7CFFFBFFB4152BFF3A3C3CFF5A" box="[1046,1062,176,195]" country="USA" lsid="urn:lsid:biocol.org:col:14792" name="Yale University" pageId="2" pageNumber="3" type="Herbarium">Y</collectionCode>
|
||
. It could be seen that in these two structures, the conformation of the docked pinyl cation is quite different (
|
||
<figureCitation id="133F2A3CFFFBFFB41443FF413DDFFF46" box="[1406,1477,203,223]" captionStart="Fig" captionStartId="5.[100,130,336,353]" captionTargetBox="[107,766,149,308]" captionTargetId="figure-640@5.[106,767,148,309]" captionTargetPageId="5" captionText="Fig. 4. Conformational differences in the pinyl cation in WT and F482Y as demonstrated by molecular dynamics simulation. The structures of WT and F482Y are superimposed. Results after 0, 1 and 2 ns of simulation are shown in a, d and c, respectively. Carbon in WT is colored in yellow and carbon in F482Y is colored in green. Oxygen is colored in red. Nitrogen is colored in blue. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)" figureDoi="http://doi.org/10.5281/zenodo.8291077" httpUri="https://zenodo.org/record/8291077/files/figure.png" pageId="2" pageNumber="3">Fig. 4A</figureCitation>
|
||
). We then performed a 2 ns molecular dynamics simulation. The results suggest more conformational changes (
|
||
<figureCitation id="133F2A3CFFFBFFB41599FE893D18FE8F" box="[1188,1282,259,279]" captionStart="Fig" captionStartId="5.[100,130,336,353]" captionTargetBox="[107,766,149,308]" captionTargetId="figure-640@5.[106,767,148,309]" captionTargetPageId="5" captionText="Fig. 4. Conformational differences in the pinyl cation in WT and F482Y as demonstrated by molecular dynamics simulation. The structures of WT and F482Y are superimposed. Results after 0, 1 and 2 ns of simulation are shown in a, d and c, respectively. Carbon in WT is colored in yellow and carbon in F482Y is colored in green. Oxygen is colored in red. Nitrogen is colored in blue. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)" figureDoi="http://doi.org/10.5281/zenodo.8291077" httpUri="https://zenodo.org/record/8291077/files/figure.png" pageId="2" pageNumber="3">Fig. 4B–C</figureCitation>
|
||
). Therefore, the extra hydroxyl group in Tyr could significantly change the conformation of the carbocation, which may explain the decreased activity and altered product profile.
|
||
</paragraph>
|
||
<paragraph id="8BBB36B9FFFBFFB4120FFE123C75FE5E" blockId="2.[818,1445,408,455]" pageId="2" pageNumber="3">
|
||
<emphasis id="B970EAABFFFBFFB4120FFE123C75FE5E" bold="true" italics="true" pageId="2" pageNumber="3">
|
||
2.4.
|
||
<collectionCode id="ED15AE7CFFFBFFB4125FFE123B74FE32" box="[866,878,408,427]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="2" pageNumber="3" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFBFFB412ADFE123B85FE32" box="[912,927,408,427]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="2" pageNumber="3" type="Herbarium">A</collectionCode>
|
||
mutation enhances the overall activity of pinene synthase without changing its product profile
|
||
</emphasis>
|
||
</paragraph>
|
||
<paragraph id="8BBB36B9FFFBFFB4126CFE663BC4FD12" blockId="2.[818,1488,492,1265]" pageId="2" pageNumber="3">
|
||
During our Ala scanning experiments, we identified a mutation
|
||
<collectionCode id="ED15AE7CFFFBFFB4120FFD823B27FD82" box="[818,829,520,539]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="2" pageNumber="3" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFBFFB41263FD823B75FD82" box="[862,879,520,539]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="2" pageNumber="3" type="Herbarium">A</collectionCode>
|
||
that enhances overall activities (
|
||
<figureCitation id="133F2A3CFFFBFFB41599FD823D19FD82" box="[1188,1283,520,539]" captionStart="Fig" captionStartId="5.[100,130,1888,1905]" captionTargetBox="[188,1399,1187,1859]" captionTargetId="figure-736@5.[187,1400,1186,1860]" captionTargetPageId="5" captionText="Fig. 5. The effect of the S491A mutation. Chromatograms in a, b and c show the GC-MS analysis of terpene production for S491A, F482A/S491A, and F482I/S491A, respectively. The x-axis is the retention time and the y-axis is the relative abundance of each species. The numbers in each peak correspond to α-pinene (1), sabinene (2) and limonene (3). d shows the overall activity and the percentage of sabinene within total products produced by F482A, F482A/S491A, F482I and F482I/S491A. The asterisk indicates P <0.05." figureDoi="http://doi.org/10.5281/zenodo.8291079" httpUri="https://zenodo.org/record/8291079/files/figure.png" pageId="2" pageNumber="3">Fig. 5A, D</figureCitation>
|
||
,
|
||
<tableCitation id="C6860302FFFBFFB41431FD823D48FD82" box="[1292,1362,520,539]" captionStart="Table 1" captionStartId="2.[100,150,1512,1528]" captionTargetPageId="2" captionText="Table 1 Major products (%±SD) of WT α-pinene synthase and enzymes with mutations." httpUri="http://table.plazi.org/id/DF7B6631FFFBFFB41159FA623B1DF98A" pageId="2" pageNumber="3" tableUuid="DF7B6631FFFBFFB41159FA623B1DF98A">Table 1</tableCitation>
|
||
). The overall activity of
|
||
<collectionCode id="ED15AE7CFFFBFFB412A2FDAE3BB0FDAE" box="[927,938,548,567]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="2" pageNumber="3" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFBFFB412F6FDAE3BC6FDAE" box="[971,988,548,567]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="2" pageNumber="3" type="Herbarium">A</collectionCode>
|
||
mutant is about 29% higher than that of the WT enzyme (
|
||
<tableCitation id="C6860302FFFBFFB412B6FDCA3BCEFDCA" box="[907,980,576,595]" captionStart="Table 1" captionStartId="2.[100,150,1512,1528]" captionTargetPageId="2" captionText="Table 1 Major products (%±SD) of WT α-pinene synthase and enzymes with mutations." httpUri="http://table.plazi.org/id/DF7B6631FFFBFFB41159FA623B1DF98A" pageId="2" pageNumber="3" tableUuid="DF7B6631FFFBFFB41159FA623B1DF98A">Table 1</tableCitation>
|
||
). Furthermore,
|
||
<collectionCode id="ED15AE7CFFFBFFB4154CFDCA3C66FDCA" box="[1137,1148,576,595]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="2" pageNumber="3" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFBFFB415A3FDCA3CB5FDCA" box="[1182,1199,576,595]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="2" pageNumber="3" type="Herbarium">A</collectionCode>
|
||
does not change the product selectivity, as more than 90% of their products remains α- pinene (
|
||
<figureCitation id="133F2A3CFFFBFFB41207FDFD3B9AFD12" box="[826,896,631,651]" captionStart="Fig" captionStartId="5.[100,130,1888,1905]" captionTargetBox="[188,1399,1187,1859]" captionTargetId="figure-736@5.[187,1400,1186,1860]" captionTargetPageId="5" captionText="Fig. 5. The effect of the S491A mutation. Chromatograms in a, b and c show the GC-MS analysis of terpene production for S491A, F482A/S491A, and F482I/S491A, respectively. The x-axis is the retention time and the y-axis is the relative abundance of each species. The numbers in each peak correspond to α-pinene (1), sabinene (2) and limonene (3). d shows the overall activity and the percentage of sabinene within total products produced by F482A, F482A/S491A, F482I and F482I/S491A. The asterisk indicates P <0.05." figureDoi="http://doi.org/10.5281/zenodo.8291079" httpUri="https://zenodo.org/record/8291079/files/figure.png" pageId="2" pageNumber="3">Fig. 5A</figureCitation>
|
||
,
|
||
<tableCitation id="C6860302FFFBFFB412B7FDFD3BCAFD12" box="[906,976,631,651]" captionStart="Table 1" captionStartId="2.[100,150,1512,1528]" captionTargetPageId="2" captionText="Table 1 Major products (%±SD) of WT α-pinene synthase and enzymes with mutations." httpUri="http://table.plazi.org/id/DF7B6631FFFBFFB41159FA623B1DF98A" pageId="2" pageNumber="3" tableUuid="DF7B6631FFFBFFB41159FA623B1DF98A">Table 1</tableCitation>
|
||
).
|
||
</paragraph>
|
||
<paragraph id="8BBB36B9FFFBFFB4126CFD193CF8FC6C" blockId="2.[818,1488,492,1265]" pageId="2" pageNumber="3">
|
||
What is the mechanism of
|
||
<collectionCode id="ED15AE7CFFFBFFB41575FD193C49FD3F" box="[1096,1107,659,678]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="2" pageNumber="3" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFBFFB41549FD193C9FFD3F" box="[1140,1157,659,678]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="2" pageNumber="3" type="Herbarium">A</collectionCode>
|
||
to enhance the enzymatic activity? Since
|
||
<collectionCode id="ED15AE7CFFFBFFB41257FD253B6FFD5B" box="[874,885,687,706]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="2" pageNumber="3" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFBFFB412AAFD253BB2FD5B" box="[919,936,687,706]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="2" pageNumber="3" type="Herbarium">A</collectionCode>
|
||
still predominantly produces pinene, we docked the pinyl cation into the active pocket of the mutant enzyme and performed a 2 ns of molecular dynamics simulation. We also performed the same dynamic simulation on the WT enzyme in complex with the pinyl cation. Our data suggests that the active site residues in
|
||
<collectionCode id="ED15AE7CFFFBFFB41581FC953CDDFCAB" box="[1212,1223,799,818]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="2" pageNumber="3" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFBFFB415D5FC953CE3FCAB" box="[1256,1273,799,818]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="2" pageNumber="3" type="Herbarium">A</collectionCode>
|
||
have a much smaller RMSD than those in the WT enzyme, indicating the mutation
|
||
<collectionCode id="ED15AE7CFFFBFFB414AFFCB13D87FCD7" box="[1426,1437,827,846]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="2" pageNumber="3" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFBFFB41483FCB13DD5FCD7" box="[1470,1487,827,846]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="2" pageNumber="3" type="Herbarium">A</collectionCode>
|
||
could cause the active pocket to be more rigid (
|
||
<figureCitation id="133F2A3CFFFBFFB41428FCDD3D45FCF3" box="[1301,1375,855,874]" captionStart="Fig" captionStartId="6.[100,130,1579,1596]" captionTargetBox="[188,1399,149,1551]" captionTargetId="figure-239@6.[187,1400,148,1552]" captionTargetPageId="6" captionText="Fig. 6. S491A mutation increases the rigidity of the active pocket when it binds the pinyl cation. RMSD values of the active site residues between WT and S491A in complex with the pinyl cation (a), terpinyl cation (b) and thujyl cation (c) are compared." figureDoi="http://doi.org/10.5281/zenodo.8291081" httpUri="https://zenodo.org/record/8291081/files/figure.png" pageId="2" pageNumber="3">Fig. 6A</figureCitation>
|
||
, STable 2). Interestingly, simulation on the WT or
|
||
<collectionCode id="ED15AE7CFFFBFFB415A3FCF93CB3FC1F" box="[1182,1193,883,902]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="2" pageNumber="3" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFBFFB415F6FCF93CC6FC1F" box="[1227,1244,883,902]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="2" pageNumber="3" type="Herbarium">A</collectionCode>
|
||
enzymes in complex with the terpinyl or thujyl cation does not show the same differences in RMSD values of active site residues (
|
||
<figureCitation id="133F2A3CFFFBFFB4156BFC203CAFFC24" box="[1110,1205,938,958]" captionStart="Fig" captionStartId="6.[100,130,1579,1596]" captionTargetBox="[188,1399,149,1551]" captionTargetId="figure-239@6.[187,1400,148,1552]" captionTargetPageId="6" captionText="Fig. 6. S491A mutation increases the rigidity of the active pocket when it binds the pinyl cation. RMSD values of the active site residues between WT and S491A in complex with the pinyl cation (a), terpinyl cation (b) and thujyl cation (c) are compared." figureDoi="http://doi.org/10.5281/zenodo.8291081" httpUri="https://zenodo.org/record/8291081/files/figure.png" pageId="2" pageNumber="3">Fig. 6B–C</figureCitation>
|
||
, STable 2). This probably indicates that the
|
||
<collectionCode id="ED15AE7CFFFBFFB412E8FC4C3BFAFC40" box="[981,992,966,985]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="2" pageNumber="3" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFBFFB4153CFC4C3C08FC40" box="[1025,1042,966,985]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="2" pageNumber="3" type="Herbarium">A</collectionCode>
|
||
mutation increases the rigidity of the active pocket only when it binds to the pinyl cation.
|
||
</paragraph>
|
||
<paragraph id="8BBB36B9FFFBFFB4126CFC743C75FB69" blockId="2.[818,1488,492,1265]" pageId="2" pageNumber="3">
|
||
To better quantify the mobility of protein structures, we reanalyzed the simulation data using MDLovoFit (
|
||
<bibRefCitation id="EF954B48FFFBFFB41597FB903D27FBB4" author="Martinez, L." box="[1194,1341,1050,1069]" pageId="2" pageNumber="3" pagination="0119264" refId="ref8102" refString="Martinez, L., 2015. Automatic identification of mobile and rigid substructures in molecular dynamics simulations and fractional structural fluctuation analysis. PloS One 10, e 0119264." type="journal article" year="2015">Martínez, 2015</bibRefCitation>
|
||
). This way we could subdivide the protein into regions with high and low mobility (
|
||
<figureCitation id="133F2A3CFFFBFFB41207FBD83B83FBFC" box="[826,921,1106,1125]" captionStart="Fig" captionStartId="7.[100,130,503,520]" captionTargetBox="[188,1399,149,475]" captionTargetId="figure-1032@7.[187,1400,148,476]" captionTargetPageId="7" captionText="Fig. 7. The RMSD profiles of the region with high (RMSD H) and low (RMSD L) mobility from the simulation of WT (a), S491A (b) and F482/S491A (c) in complex with the pinyl cation." figureDoi="http://doi.org/10.5281/zenodo.8291083" httpUri="https://zenodo.org/record/8291083/files/figure.png" pageId="2" pageNumber="3">Fig. 7A–C</figureCitation>
|
||
, STable 3). Interestingly, in
|
||
<collectionCode id="ED15AE7CFFFBFFB4159AFBD83CA8FBFC" box="[1191,1202,1106,1125]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="2" pageNumber="3" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFBFFB415E8FBD83CFCFBFC" box="[1237,1254,1106,1125]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="2" pageNumber="3" type="Herbarium">A</collectionCode>
|
||
, fewer residues are classified as of high mobility than residues in WT enzymes (59% vs 63%). Furthermore, for residues classified with the highly mobile subset, those in
|
||
<collectionCode id="ED15AE7CFFFBFFB41276FB2F3B4CFB21" box="[843,854,1189,1208]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="2" pageNumber="3" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFBFFB41245FB2F3B93FB21" box="[888,905,1189,1208]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="2" pageNumber="3" type="Herbarium">A</collectionCode>
|
||
have lower average RMSD than those in WT proteins (
|
||
<quantity id="4CFC9B5CFFFBFFB414A5FB2F3B4AFB4C" metricMagnitude="-10" metricUnit="m" metricValue="2.53" pageId="2" pageNumber="3" unit="nm" value="0.253">0.253 nm</quantity>
|
||
vs.
|
||
<quantity id="4CFC9B5CFFFBFFB41248FB4B3BC8FB4D" box="[885,978,1217,1236]" metricMagnitude="-10" metricUnit="m" metricValue="2.78" pageId="2" pageNumber="3" unit="nm" value="0.278">0.278 nm</quantity>
|
||
). Therefore, there is a slight but significant increase in rigidity in
|
||
<collectionCode id="ED15AE7CFFFBFFB412ABFB573BBBFB69" box="[918,929,1245,1264]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="2" pageNumber="3" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFBFFB412FEFB573BCEFB69" box="[963,980,1245,1264]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="2" pageNumber="3" type="Herbarium">A</collectionCode>
|
||
mutant protein.
|
||
</paragraph>
|
||
<paragraph id="8BBB36B9FFFBFFB4120FFA943BACFAD4" blockId="2.[818,1468,1310,1357]" pageId="2" pageNumber="3">
|
||
<emphasis id="B970EAABFFFBFFB4120FFA943BACFAD4" bold="true" italics="true" pageId="2" pageNumber="3">
|
||
2.5. Combination of
|
||
<collectionCode id="ED15AE7CFFFBFFB412CEFA943BE5FAA8" box="[1011,1023,1310,1329]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="2" pageNumber="3" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFBFFB4151CFA943C2AFAA8" box="[1057,1072,1310,1329]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="2" pageNumber="3" type="Herbarium">A</collectionCode>
|
||
with
|
||
<collectionCode id="ED15AE7CFFFBFFB41559FA943C69FAA8" box="[1124,1139,1310,1329]" country="USA" lsid="urn:lsid:biocol.org:col:15707" name="Field Museum of Natural History, Botany Department" pageId="2" pageNumber="3" type="Herbarium">F</collectionCode>
|
||
482 mutants has mixed results in the overall activity
|
||
</emphasis>
|
||
</paragraph>
|
||
<paragraph id="8BBB36B9FFFBFFB5126CFAF8390BFA6F" blockId="2.[818,1488,1394,1469]" lastBlockId="3.[100,771,1312,1973]" lastPageId="3" lastPageNumber="4" pageId="2" pageNumber="3">
|
||
Since all four sabinene synthase mutants show some decreases in the catalytic activity, we wondered whether we may restore their activities to that of the WT pinene synthase by combing with
|
||
<collectionCode id="ED15AE7CFFFBFFB41431FA203D0DFA24" box="[1292,1303,1450,1469]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="2" pageNumber="3" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFBFFB41406FA203D57FA24" box="[1339,1357,1450,1469]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="2" pageNumber="3" type="Herbarium">A</collectionCode>
|
||
. We produced double mutants
|
||
<collectionCode id="ED15AE7CFFFAFFB5103DFAAA3917FAAA" box="[256,269,1312,1331]" country="USA" lsid="urn:lsid:biocol.org:col:15707" name="Field Museum of Natural History, Botany Department" pageId="3" pageNumber="4" type="Herbarium">F</collectionCode>
|
||
482
|
||
<collectionCode id="ED15AE7CFFFAFFB5100CFAAA3958FAAA" box="[305,322,1312,1331]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
|
||
/
|
||
<collectionCode id="ED15AE7CFFFAFFB51074FAAA394EFAAA" box="[329,340,1312,1331]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="3" pageNumber="4" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFAFFB51048FAAA399CFAAA" box="[373,390,1312,1331]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
|
||
and
|
||
<collectionCode id="ED15AE7CFFFAFFB51084FAAA39DCFAAA" box="[441,454,1312,1331]" country="USA" lsid="urn:lsid:biocol.org:col:15707" name="Field Museum of Natural History, Botany Department" pageId="3" pageNumber="4" type="Herbarium">F</collectionCode>
|
||
482
|
||
<collectionCode id="ED15AE7CFFFAFFB510D6FAAA39E9FAAA" box="[491,499,1312,1331]" country="Romania" lsid="urn:lsid:biocol.org:col:14415" name="&quot;Alexandru Ioan Cuza&quot; University" pageId="3" pageNumber="4" type="Herbarium">I</collectionCode>
|
||
/
|
||
<collectionCode id="ED15AE7CFFFAFFB510C7FAAA3A1FFAAA" box="[506,517,1312,1331]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="3" pageNumber="4" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFAFFB51315FAAA3A23FAAA" box="[552,569,1312,1331]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
|
||
. Both seem to retain the same product selectivity of the original single mutants, though
|
||
<collectionCode id="ED15AE7CFFFAFFB51159FAD2386BFAF2" box="[100,113,1368,1387]" country="USA" lsid="urn:lsid:biocol.org:col:15707" name="Field Museum of Natural History, Botany Department" pageId="3" pageNumber="4" type="Herbarium">F</collectionCode>
|
||
482
|
||
<collectionCode id="ED15AE7CFFFAFFB511A8FAD238BCFAF2" box="[149,166,1368,1387]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
|
||
/
|
||
<collectionCode id="ED15AE7CFFFAFFB51190FAD238A2FAF2" box="[173,184,1368,1387]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="3" pageNumber="4" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFAFFB511E4FAD238F0FAF2" box="[217,234,1368,1387]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
|
||
shows an insignificant decrease in sabinene production (P =0.39;
|
||
<figureCitation id="133F2A3CFFFAFFB511F4FAFE393FFA1E" box="[201,293,1396,1415]" captionStart="Fig" captionStartId="5.[100,130,1888,1905]" captionTargetBox="[188,1399,1187,1859]" captionTargetId="figure-736@5.[187,1400,1186,1860]" captionTargetPageId="5" captionText="Fig. 5. The effect of the S491A mutation. Chromatograms in a, b and c show the GC-MS analysis of terpene production for S491A, F482A/S491A, and F482I/S491A, respectively. The x-axis is the retention time and the y-axis is the relative abundance of each species. The numbers in each peak correspond to α-pinene (1), sabinene (2) and limonene (3). d shows the overall activity and the percentage of sabinene within total products produced by F482A, F482A/S491A, F482I and F482I/S491A. The asterisk indicates P <0.05." figureDoi="http://doi.org/10.5281/zenodo.8291079" httpUri="https://zenodo.org/record/8291079/files/figure.png" pageId="3" pageNumber="4">Fig. 5B–C</figureCitation>
|
||
,
|
||
<tableCitation id="C6860302FFFAFFB51010FAFE3969FA1E" box="[301,371,1396,1415]" captionStart="Table 1" captionStartId="2.[100,150,1512,1528]" captionTargetPageId="2" captionText="Table 1 Major products (%±SD) of WT α-pinene synthase and enzymes with mutations." httpUri="http://table.plazi.org/id/DF7B6631FFFBFFB41159FA623B1DF98A" pageId="3" pageNumber="4" tableUuid="DF7B6631FFFBFFB41159FA623B1DF98A">Table 1</tableCitation>
|
||
). In overall activities,
|
||
<collectionCode id="ED15AE7CFFFAFFB51378FAFE3A48FA1E" box="[581,594,1396,1415]" country="USA" lsid="urn:lsid:biocol.org:col:15707" name="Field Museum of Natural History, Botany Department" pageId="3" pageNumber="4" type="Herbarium">F</collectionCode>
|
||
482
|
||
<collectionCode id="ED15AE7CFFFAFFB5134BFAFE3A67FA1E" box="[630,637,1396,1415]" country="Romania" lsid="urn:lsid:biocol.org:col:14415" name="&quot;Alexandru Ioan Cuza&quot; University" pageId="3" pageNumber="4" type="Herbarium">I</collectionCode>
|
||
/
|
||
<collectionCode id="ED15AE7CFFFAFFB513B8FAFE3A8AFA1E" box="[645,656,1396,1415]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="3" pageNumber="4" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFAFFB5138FFAFE3AD9FA1E" box="[690,707,1396,1415]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
|
||
shows an insignificant increase in activity (P =0.27), while
|
||
<collectionCode id="ED15AE7CFFFAFFB5136AFA1A3A7EFA3A" box="[599,612,1424,1443]" country="USA" lsid="urn:lsid:biocol.org:col:15707" name="Field Museum of Natural History, Botany Department" pageId="3" pageNumber="4" type="Herbarium">F</collectionCode>
|
||
482
|
||
<collectionCode id="ED15AE7CFFFAFFB513B5FA1A3A83FA3A" box="[648,665,1424,1443]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
|
||
/
|
||
<collectionCode id="ED15AE7CFFFAFFB5139DFA1A3AB1FA3A" box="[672,683,1424,1443]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="3" pageNumber="4" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFAFFB513F1FA1A3AC7FA3A" box="[716,733,1424,1443]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
|
||
has a significant decrease in activity (P
|
||
<emphasis id="B970EAABFFFAFFB51092FA2639A5FA26" box="[431,447,1452,1471]" italics="true" pageId="3" pageNumber="4"><</emphasis>
|
||
0.05;
|
||
<figureCitation id="133F2A3CFFFAFFB510C7FA263A25FA26" box="[506,575,1451,1471]" captionStart="Fig" captionStartId="5.[100,130,1888,1905]" captionTargetBox="[188,1399,1187,1859]" captionTargetId="figure-736@5.[187,1400,1186,1860]" captionTargetPageId="5" captionText="Fig. 5. The effect of the S491A mutation. Chromatograms in a, b and c show the GC-MS analysis of terpene production for S491A, F482A/S491A, and F482I/S491A, respectively. The x-axis is the retention time and the y-axis is the relative abundance of each species. The numbers in each peak correspond to α-pinene (1), sabinene (2) and limonene (3). d shows the overall activity and the percentage of sabinene within total products produced by F482A, F482A/S491A, F482I and F482I/S491A. The asterisk indicates P <0.05." figureDoi="http://doi.org/10.5281/zenodo.8291079" httpUri="https://zenodo.org/record/8291079/files/figure.png" pageId="3" pageNumber="4">Fig. 5D</figureCitation>
|
||
,
|
||
<tableCitation id="C6860302FFFAFFB51375FA263A97FA26" box="[584,653,1452,1471]" captionStart="Table 1" captionStartId="2.[100,150,1512,1528]" captionTargetPageId="2" captionText="Table 1 Major products (%±SD) of WT α-pinene synthase and enzymes with mutations." httpUri="http://table.plazi.org/id/DF7B6631FFFBFFB41159FA623B1DF98A" pageId="3" pageNumber="4" tableUuid="DF7B6631FFFBFFB41159FA623B1DF98A">Table 1</tableCitation>
|
||
). Therefore, it seems that the effect of combining two mutants is more complicated than we expected.
|
||
</paragraph>
|
||
<caption id="DF7B6631FFFBFFB41159FA623B1DF98A" ID-Table-UUID="DF7B6631FFFBFFB41159FA623B1DF98A" httpUri="http://table.plazi.org/id/DF7B6631FFFBFFB41159FA623B1DF98A" pageId="2" pageNumber="3" startId="2.[100,150,1512,1528]" targetBox="[116,1472,1575,1969]" targetIsTable="true" targetPageId="2" targetType="table">
|
||
<paragraph id="8BBB36B9FFFBFFB41159FA6238BDFA60" blockId="2.[100,775,1512,1556]" box="[100,167,1512,1529]" pageId="2" pageNumber="3">
|
||
<emphasis id="B970EAABFFFBFFB41159FA6238BDFA60" bold="true" box="[100,167,1512,1529]" pageId="2" pageNumber="3">Table 1</emphasis>
|
||
</paragraph>
|
||
<paragraph id="8BBB36B9FFFBFFB41159F9883B1DF98A" blockId="2.[100,775,1512,1556]" box="[100,775,1537,1556]" pageId="2" pageNumber="3">Major products (%±SD) of WT α- pinene synthase and enzymes with mutations.</paragraph>
|
||
</caption>
|
||
<paragraph id="8BBB36B9FFFBFFB41149F9AD3DB7F829" pageId="2" pageNumber="3">
|
||
<table id="F904C419FFFB00491149F9AD3DDAF828" box="[116,1472,1575,1969]" gridcols="8" gridrows="16" pageId="2" pageNumber="3">
|
||
<tr id="353434FBFFFB00491149F9AD3DDAF9AC" box="[116,1472,1575,1589]" gridrow="0" pageId="2" pageNumber="3" rowspan-3="1" rowspan-4="1" rowspan-5="1" rowspan-6="1" rowspan-7="1">
|
||
<th id="76E55D87FFFB00491149F9AD38C5F9AC" box="[116,223,1575,1589]" gridcol="0" gridrow="0" pageId="2" pageNumber="3">Enzymes</th>
|
||
<th id="76E55D87FFFB00491015F9AD3A22F9AC" box="[296,568,1575,1589]" colspan="2" colspanRight="1" gridcol="1" gridrow="0" pageId="2" pageNumber="3">Relative Proportion of Products (%)</th>
|
||
</tr>
|
||
<tr id="353434FBFFFB00491149F9C23DDAF9C0" box="[116,1472,1608,1625]" gridrow="1" pageId="2" pageNumber="3" rowspan-0="1">
|
||
<td id="76E55D87FFFB00491015F9C23996F9C0" box="[296,396,1608,1625]" gridcol="1" gridrow="1" pageId="2" pageNumber="3">Unknown</td>
|
||
<td id="76E55D87FFFB004910E8F9C23A22F9C0" box="[469,568,1608,1625]" gridcol="2" gridrow="1" pageId="2" pageNumber="3">α- pinene</td>
|
||
<td id="76E55D87FFFB004913BFF9C23AEAF9C0" box="[642,752,1608,1625]" gridcol="3" gridrow="1" pageId="2" pageNumber="3">sabinene</td>
|
||
<td id="76E55D87FFFB00491204F9C23BBCF9C0" box="[825,934,1608,1625]" gridcol="4" gridrow="1" pageId="2" pageNumber="3">limonene</td>
|
||
<td id="76E55D87FFFB004912D2F9C23C47F9C0" box="[1007,1117,1608,1625]" gridcol="5" gridrow="1" pageId="2" pageNumber="3">Unknown</td>
|
||
<td id="76E55D87FFFB0049159BF9C23D1AF9C0" box="[1190,1280,1608,1625]" gridcol="6" gridrow="1" pageId="2" pageNumber="3">Unknown</td>
|
||
<td id="76E55D87FFFB00491474F9C23DDAF9C0" box="[1353,1472,1608,1625]" gridcol="7" gridrow="1" pageId="2" pageNumber="3">Activity(% WT)</td>
|
||
</tr>
|
||
<tr id="353434FBFFFB00491149F9E63DDAF9E2" box="[116,1472,1644,1659]" gridrow="2" pageId="2" pageNumber="3" rowspan-0="1" rowspan-7="1">
|
||
<td id="76E55D87FFFB00491015F9E63996F9E2" box="[296,396,1644,1659]" gridcol="1" gridrow="2" pageId="2" pageNumber="3">RT = 4.52</td>
|
||
<td id="76E55D87FFFB004910E8F9E63A22F9E2" box="[469,568,1644,1659]" gridcol="2" gridrow="2" pageId="2" pageNumber="3">RT = 7.05</td>
|
||
<td id="76E55D87FFFB004913BFF9E63AEAF9E2" box="[642,752,1644,1659]" gridcol="3" gridrow="2" pageId="2" pageNumber="3">RT = 8.55</td>
|
||
<td id="76E55D87FFFB00491204F9E63BBCF9E2" box="[825,934,1644,1659]" gridcol="4" gridrow="2" pageId="2" pageNumber="3">RT = 11.05</td>
|
||
<td id="76E55D87FFFB004912D2F9E63C47F9E2" box="[1007,1117,1644,1659]" gridcol="5" gridrow="2" pageId="2" pageNumber="3">RT = 11.58</td>
|
||
<td id="76E55D87FFFB0049159BF9E63D1AF9E2" box="[1190,1280,1644,1659]" gridcol="6" gridrow="2" pageId="2" pageNumber="3">RT = 12.72</td>
|
||
</tr>
|
||
<tr id="353434FBFFFB00491149F9053DDAF907" box="[116,1472,1679,1694]" gridrow="3" pageId="2" pageNumber="3">
|
||
<th id="76E55D87FFFB00491149F90538C5F907" box="[116,223,1679,1694]" gridcol="0" gridrow="3" pageId="2" pageNumber="3">PS-WT</th>
|
||
<td id="76E55D87FFFB00491015F9053996F907" box="[296,396,1679,1694]" gridcol="1" gridrow="3" pageId="2" pageNumber="3">0.31 ± 0.02</td>
|
||
<td id="76E55D87FFFB004910E8F9053A22F907" box="[469,568,1679,1694]" gridcol="2" gridrow="3" pageId="2" pageNumber="3">97.86 ± 0.21</td>
|
||
<td id="76E55D87FFFB004913BFF9053AEAF907" box="[642,752,1679,1694]" gridcol="3" gridrow="3" pageId="2" pageNumber="3">0.96 ± 0.00</td>
|
||
<td id="76E55D87FFFB00491204F9053BBCF907" box="[825,934,1679,1694]" gridcol="4" gridrow="3" pageId="2" pageNumber="3">0.26 ± 0.01</td>
|
||
<td id="76E55D87FFFB004912D2F9053C47F907" box="[1007,1117,1679,1694]" gridcol="5" gridrow="3" pageId="2" pageNumber="3">0.54 ± 0.05</td>
|
||
<td id="76E55D87FFFB0049159BF9053D1AF907" box="[1190,1280,1679,1694]" gridcol="6" gridrow="3" pageId="2" pageNumber="3">0.07 ± 0.00</td>
|
||
<td id="76E55D87FFFB00491474F9053DDAF907" box="[1353,1472,1679,1694]" gridcol="7" gridrow="3" pageId="2" pageNumber="3">100</td>
|
||
</tr>
|
||
<tr id="353434FBFFFB00491149F92C3DDAF92C" box="[116,1472,1702,1717]" gridrow="4" pageId="2" pageNumber="3">
|
||
<th id="76E55D87FFFB00491149F92C38C5F92C" box="[116,223,1702,1717]" gridcol="0" gridrow="4" pageId="2" pageNumber="3">F482A</th>
|
||
<td id="76E55D87FFFB00491015F92C3996F92C" box="[296,396,1702,1717]" gridcol="1" gridrow="4" pageId="2" pageNumber="3">0.05 ± 0.00</td>
|
||
<td id="76E55D87FFFB004910E8F92C3A22F92C" box="[469,568,1702,1717]" gridcol="2" gridrow="4" pageId="2" pageNumber="3">4.43 ± 0.03</td>
|
||
<td id="76E55D87FFFB004913BFF92C3AEAF92C" box="[642,752,1702,1717]" gridcol="3" gridrow="4" pageId="2" pageNumber="3">93.21 ± 0.06</td>
|
||
<td id="76E55D87FFFB00491204F92C3BBCF92C" box="[825,934,1702,1717]" gridcol="4" gridrow="4" pageId="2" pageNumber="3">1.59 ± 0.05</td>
|
||
<td id="76E55D87FFFB004912D2F92C3C47F92C" box="[1007,1117,1702,1717]" gridcol="5" gridrow="4" pageId="2" pageNumber="3">0.72 ± 0.03</td>
|
||
<td id="76E55D87FFFB0049159BF92C3D1AF92C" box="[1190,1280,1702,1717]" gridcol="6" gridrow="4" pageId="2" pageNumber="3">0.00 ± 0.00</td>
|
||
<td id="76E55D87FFFB00491474F92C3DDAF92C" box="[1353,1472,1702,1717]" gridcol="7" gridrow="4" pageId="2" pageNumber="3">46.63 ± 4.47</td>
|
||
</tr>
|
||
<tr id="353434FBFFFB00491149F9373DDAF955" box="[116,1472,1725,1740]" gridrow="5" pageId="2" pageNumber="3">
|
||
<th id="76E55D87FFFB00491149F93738C5F955" box="[116,223,1725,1740]" gridcol="0" gridrow="5" pageId="2" pageNumber="3">S491A</th>
|
||
<td id="76E55D87FFFB00491015F9373996F955" box="[296,396,1725,1740]" gridcol="1" gridrow="5" pageId="2" pageNumber="3">0.46 ± 0.01</td>
|
||
<td id="76E55D87FFFB004910E8F9373A22F955" box="[469,568,1725,1740]" gridcol="2" gridrow="5" pageId="2" pageNumber="3">97.28 ± 0.09</td>
|
||
<td id="76E55D87FFFB004913BFF9373AEAF955" box="[642,752,1725,1740]" gridcol="3" gridrow="5" pageId="2" pageNumber="3">1.14 ± 0.00</td>
|
||
<td id="76E55D87FFFB00491204F9373BBCF955" box="[825,934,1725,1740]" gridcol="4" gridrow="5" pageId="2" pageNumber="3">0.12 ± 0.00</td>
|
||
<td id="76E55D87FFFB004912D2F9373C47F955" box="[1007,1117,1725,1740]" gridcol="5" gridrow="5" pageId="2" pageNumber="3">0.99 ± 0.06</td>
|
||
<td id="76E55D87FFFB0049159BF9373D1AF955" box="[1190,1280,1725,1740]" gridcol="6" gridrow="5" pageId="2" pageNumber="3">0.00 ± 0.00</td>
|
||
<td id="76E55D87FFFB00491474F9373DDAF955" box="[1353,1472,1725,1740]" gridcol="7" gridrow="5" pageId="2" pageNumber="3">128.90 ± 4.19</td>
|
||
</tr>
|
||
<tr id="353434FBFFFB00491149F95E3DDAF97A" box="[116,1472,1748,1763]" gridrow="6" pageId="2" pageNumber="3">
|
||
<th id="76E55D87FFFB00491149F95E38C5F97A" box="[116,223,1748,1763]" gridcol="0" gridrow="6" pageId="2" pageNumber="3">F482I</th>
|
||
<td id="76E55D87FFFB00491015F95E3996F97A" box="[296,396,1748,1763]" gridcol="1" gridrow="6" pageId="2" pageNumber="3">1.27 ± 0.02</td>
|
||
<td id="76E55D87FFFB004910E8F95E3A22F97A" box="[469,568,1748,1763]" gridcol="2" gridrow="6" pageId="2" pageNumber="3">3.17 ± 0.08</td>
|
||
<td id="76E55D87FFFB004913BFF95E3AEAF97A" box="[642,752,1748,1763]" gridcol="3" gridrow="6" pageId="2" pageNumber="3">92.35 ± 0.21</td>
|
||
<td id="76E55D87FFFB00491204F95E3BBCF97A" box="[825,934,1748,1763]" gridcol="4" gridrow="6" pageId="2" pageNumber="3">0.66 ± 0.09</td>
|
||
<td id="76E55D87FFFB004912D2F95E3C47F97A" box="[1007,1117,1748,1763]" gridcol="5" gridrow="6" pageId="2" pageNumber="3">2.33 ± 0.08</td>
|
||
<td id="76E55D87FFFB0049159BF95E3D1AF97A" box="[1190,1280,1748,1763]" gridcol="6" gridrow="6" pageId="2" pageNumber="3">0.23 ± 0.00</td>
|
||
<td id="76E55D87FFFB00491474F95E3DDAF97A" box="[1353,1472,1748,1763]" gridcol="7" gridrow="6" pageId="2" pageNumber="3">18.36 ± 2.51</td>
|
||
</tr>
|
||
<tr id="353434FBFFFB00491149F9613DDAF963" box="[116,1472,1771,1786]" gridrow="7" pageId="2" pageNumber="3">
|
||
<th id="76E55D87FFFB00491149F96138C5F963" box="[116,223,1771,1786]" gridcol="0" gridrow="7" pageId="2" pageNumber="3">F482L</th>
|
||
<td id="76E55D87FFFB00491015F9613996F963" box="[296,396,1771,1786]" gridcol="1" gridrow="7" pageId="2" pageNumber="3">0.96 ± 0.06</td>
|
||
<td id="76E55D87FFFB004910E8F9613A22F963" box="[469,568,1771,1786]" gridcol="2" gridrow="7" pageId="2" pageNumber="3">2.03 ± 0.03</td>
|
||
<td id="76E55D87FFFB004913BFF9613AEAF963" box="[642,752,1771,1786]" gridcol="3" gridrow="7" pageId="2" pageNumber="3">94.26 ± 0.30</td>
|
||
<td id="76E55D87FFFB00491204F9613BBCF963" box="[825,934,1771,1786]" gridcol="4" gridrow="7" pageId="2" pageNumber="3">0.31 ± 0.01</td>
|
||
<td id="76E55D87FFFB004912D2F9613C47F963" box="[1007,1117,1771,1786]" gridcol="5" gridrow="7" pageId="2" pageNumber="3">1.91 ± 0.17</td>
|
||
<td id="76E55D87FFFB0049159BF9613D1AF963" box="[1190,1280,1771,1786]" gridcol="6" gridrow="7" pageId="2" pageNumber="3">0.29 ± 0.01</td>
|
||
<td id="76E55D87FFFB00491474F9613DDAF963" box="[1353,1472,1771,1786]" gridcol="7" gridrow="7" pageId="2" pageNumber="3">49.98 ± 39.78</td>
|
||
</tr>
|
||
<tr id="353434FBFFFB00491149F8883DDAF888" box="[116,1472,1794,1809]" gridrow="8" pageId="2" pageNumber="3">
|
||
<th id="76E55D87FFFB00491149F88838C5F888" box="[116,223,1794,1809]" gridcol="0" gridrow="8" pageId="2" pageNumber="3">F482V</th>
|
||
<td id="76E55D87FFFB00491015F8883996F888" box="[296,396,1794,1809]" gridcol="1" gridrow="8" pageId="2" pageNumber="3">1.03 ± 0.01</td>
|
||
<td id="76E55D87FFFB004910E8F8883A22F888" box="[469,568,1794,1809]" gridcol="2" gridrow="8" pageId="2" pageNumber="3">2.12 ± 0.03</td>
|
||
<td id="76E55D87FFFB004913BFF8883AEAF888" box="[642,752,1794,1809]" gridcol="3" gridrow="8" pageId="2" pageNumber="3">94.51 ± 0.07</td>
|
||
<td id="76E55D87FFFB00491204F8883BBCF888" box="[825,934,1794,1809]" gridcol="4" gridrow="8" pageId="2" pageNumber="3">0.30 ± 0.03</td>
|
||
<td id="76E55D87FFFB004912D2F8883C47F888" box="[1007,1117,1794,1809]" gridcol="5" gridrow="8" pageId="2" pageNumber="3">1.82 ± 0.06</td>
|
||
<td id="76E55D87FFFB0049159BF8883D1AF888" box="[1190,1280,1794,1809]" gridcol="6" gridrow="8" pageId="2" pageNumber="3">0.23 ± 0.00</td>
|
||
<td id="76E55D87FFFB00491474F8883DDAF888" box="[1353,1472,1794,1809]" gridcol="7" gridrow="8" pageId="2" pageNumber="3">29.50 ± 12.39</td>
|
||
</tr>
|
||
<tr id="353434FBFFFB00491149F8923DDAF8BE" box="[116,1472,1816,1831]" gridrow="9" pageId="2" pageNumber="3">
|
||
<th id="76E55D87FFFB00491149F89238C5F8BE" box="[116,223,1816,1831]" gridcol="0" gridrow="9" pageId="2" pageNumber="3">F482T</th>
|
||
<td id="76E55D87FFFB00491015F8923996F8BE" box="[296,396,1816,1831]" gridcol="1" gridrow="9" pageId="2" pageNumber="3">0.00 ± 0.00</td>
|
||
<td id="76E55D87FFFB004910E8F8923A22F8BE" box="[469,568,1816,1831]" gridcol="2" gridrow="9" pageId="2" pageNumber="3">3.53 ± 0.00</td>
|
||
<td id="76E55D87FFFB004913BFF8923AEAF8BE" box="[642,752,1816,1831]" gridcol="3" gridrow="9" pageId="2" pageNumber="3">93.91 ± 0.00</td>
|
||
<td id="76E55D87FFFB00491204F8923BBCF8BE" box="[825,934,1816,1831]" gridcol="4" gridrow="9" pageId="2" pageNumber="3">1.64 ± 0.04</td>
|
||
<td id="76E55D87FFFB004912D2F8923C47F8BE" box="[1007,1117,1816,1831]" gridcol="5" gridrow="9" pageId="2" pageNumber="3">0.92 ± 0.05</td>
|
||
<td id="76E55D87FFFB0049159BF8923D1AF8BE" box="[1190,1280,1816,1831]" gridcol="6" gridrow="9" pageId="2" pageNumber="3">0.00 ± 0.00</td>
|
||
<td id="76E55D87FFFB00491474F8923DDAF8BE" box="[1353,1472,1816,1831]" gridcol="7" gridrow="9" pageId="2" pageNumber="3">52.69 ± 3.17</td>
|
||
</tr>
|
||
<tr id="353434FBFFFB00491149F8BA3DDAF8A7" box="[116,1472,1840,1854]" gridrow="10" pageId="2" pageNumber="3">
|
||
<th id="76E55D87FFFB00491149F8BA38C5F8A7" box="[116,223,1840,1854]" gridcol="0" gridrow="10" pageId="2" pageNumber="3">F482Y</th>
|
||
<td id="76E55D87FFFB00491015F8BA3996F8A7" box="[296,396,1840,1854]" gridcol="1" gridrow="10" pageId="2" pageNumber="3">0.00</td>
|
||
<td id="76E55D87FFFB004910E8F8BA3A22F8A7" box="[469,568,1840,1854]" gridcol="2" gridrow="10" pageId="2" pageNumber="3">2.71</td>
|
||
<td id="76E55D87FFFB004913BFF8BA3AEAF8A7" box="[642,752,1840,1854]" gridcol="3" gridrow="10" pageId="2" pageNumber="3">31.00</td>
|
||
<td id="76E55D87FFFB00491204F8BA3BBCF8A7" box="[825,934,1840,1854]" gridcol="4" gridrow="10" pageId="2" pageNumber="3">63.61</td>
|
||
<td id="76E55D87FFFB004912D2F8BA3C47F8A7" box="[1007,1117,1840,1854]" gridcol="5" gridrow="10" pageId="2" pageNumber="3">2.68</td>
|
||
<td id="76E55D87FFFB0049159BF8BA3D1AF8A7" box="[1190,1280,1840,1854]" gridcol="6" gridrow="10" pageId="2" pageNumber="3">0.00</td>
|
||
<td id="76E55D87FFFB00491474F8BA3DDAF8A7" box="[1353,1472,1840,1854]" gridcol="7" gridrow="10" pageId="2" pageNumber="3">0.62</td>
|
||
</tr>
|
||
<tr id="353434FBFFFB00491149F8CC3DDAF8CC" box="[116,1472,1862,1877]" gridrow="11" pageId="2" pageNumber="3">
|
||
<th id="76E55D87FFFB00491149F8CC38C5F8CC" box="[116,223,1862,1877]" gridcol="0" gridrow="11" pageId="2" pageNumber="3">F482R</th>
|
||
<td id="76E55D87FFFB00491015F8CC3996F8CC" box="[296,396,1862,1877]" gridcol="1" gridrow="11" pageId="2" pageNumber="3">17.62 ± 6.21</td>
|
||
<td id="76E55D87FFFB004910E8F8CC3A22F8CC" box="[469,568,1862,1877]" gridcol="2" gridrow="11" pageId="2" pageNumber="3">0.00 ± 0.00</td>
|
||
<td id="76E55D87FFFB004913BFF8CC3AEAF8CC" box="[642,752,1862,1877]" gridcol="3" gridrow="11" pageId="2" pageNumber="3">18.96 ± 7.19</td>
|
||
<td id="76E55D87FFFB00491204F8CC3BBCF8CC" box="[825,934,1862,1877]" gridcol="4" gridrow="11" pageId="2" pageNumber="3">31.04 ± 19.27</td>
|
||
<td id="76E55D87FFFB004912D2F8CC3C47F8CC" box="[1007,1117,1862,1877]" gridcol="5" gridrow="11" pageId="2" pageNumber="3">27.33 ± 14.94</td>
|
||
<td id="76E55D87FFFB0049159BF8CC3D1AF8CC" box="[1190,1280,1862,1877]" gridcol="6" gridrow="11" pageId="2" pageNumber="3">5.05 ± 0.51</td>
|
||
<td id="76E55D87FFFB00491474F8CC3DDAF8CC" box="[1353,1472,1862,1877]" gridcol="7" gridrow="11" pageId="2" pageNumber="3">1.28 ± 0.01</td>
|
||
</tr>
|
||
<tr id="353434FBFFFB00491149F8D73DDAF8F5" box="[116,1472,1885,1900]" gridrow="12" pageId="2" pageNumber="3">
|
||
<th id="76E55D87FFFB00491149F8D738C5F8F5" box="[116,223,1885,1900]" gridcol="0" gridrow="12" pageId="2" pageNumber="3">F482W</th>
|
||
<td id="76E55D87FFFB00491015F8D73996F8F5" box="[296,396,1885,1900]" gridcol="1" gridrow="12" pageId="2" pageNumber="3">16.32 ± 5.33</td>
|
||
<td id="76E55D87FFFB004910E8F8D73A22F8F5" box="[469,568,1885,1900]" gridcol="2" gridrow="12" pageId="2" pageNumber="3">0.00 ± 0.00</td>
|
||
<td id="76E55D87FFFB004913BFF8D73AEAF8F5" box="[642,752,1885,1900]" gridcol="3" gridrow="12" pageId="2" pageNumber="3">29.92 ± 17.90</td>
|
||
<td id="76E55D87FFFB00491204F8D73BBCF8F5" box="[825,934,1885,1900]" gridcol="4" gridrow="12" pageId="2" pageNumber="3">21.67 ± 6.84</td>
|
||
<td id="76E55D87FFFB004912D2F8D73C47F8F5" box="[1007,1117,1885,1900]" gridcol="5" gridrow="12" pageId="2" pageNumber="3">27.35 ± 14.96</td>
|
||
<td id="76E55D87FFFB0049159BF8D73D1AF8F5" box="[1190,1280,1885,1900]" gridcol="6" gridrow="12" pageId="2" pageNumber="3">4.74 ± 0.45</td>
|
||
<td id="76E55D87FFFB00491474F8D73DDAF8F5" box="[1353,1472,1885,1900]" gridcol="7" gridrow="12" pageId="2" pageNumber="3">1.54 ± 0.02</td>
|
||
</tr>
|
||
<tr id="353434FBFFFB00491149F8FE3DDAF81A" box="[116,1472,1908,1923]" gridrow="13" pageId="2" pageNumber="3">
|
||
<th id="76E55D87FFFB00491149F8FE38C5F81A" box="[116,223,1908,1923]" gridcol="0" gridrow="13" pageId="2" pageNumber="3">I335A</th>
|
||
<td id="76E55D87FFFB00491015F8FE3996F81A" box="[296,396,1908,1923]" gridcol="1" gridrow="13" pageId="2" pageNumber="3">3.52 ± 0.02</td>
|
||
<td id="76E55D87FFFB004910E8F8FE3A22F81A" box="[469,568,1908,1923]" gridcol="2" gridrow="13" pageId="2" pageNumber="3">78.00 ± 0.03</td>
|
||
<td id="76E55D87FFFB004913BFF8FE3AEAF81A" box="[642,752,1908,1923]" gridcol="3" gridrow="13" pageId="2" pageNumber="3">12.94 ± 0.00</td>
|
||
<td id="76E55D87FFFB00491204F8FE3BBCF81A" box="[825,934,1908,1923]" gridcol="4" gridrow="13" pageId="2" pageNumber="3">0.57 ± 0.02</td>
|
||
<td id="76E55D87FFFB004912D2F8FE3C47F81A" box="[1007,1117,1908,1923]" gridcol="5" gridrow="13" pageId="2" pageNumber="3">4.28 ± 0.03</td>
|
||
<td id="76E55D87FFFB0049159BF8FE3D1AF81A" box="[1190,1280,1908,1923]" gridcol="6" gridrow="13" pageId="2" pageNumber="3">0.67 ± 0.00</td>
|
||
<td id="76E55D87FFFB00491474F8FE3DDAF81A" box="[1353,1472,1908,1923]" gridcol="7" gridrow="13" pageId="2" pageNumber="3">54.36 ± 2.17</td>
|
||
</tr>
|
||
<tr id="353434FBFFFB00491149F8013DDAF803" box="[116,1472,1931,1946]" gridrow="14" pageId="2" pageNumber="3">
|
||
<th id="76E55D87FFFB00491149F80138C5F803" box="[116,223,1931,1946]" gridcol="0" gridrow="14" pageId="2" pageNumber="3">F482A/S491A</th>
|
||
<td id="76E55D87FFFB00491015F8013996F803" box="[296,396,1931,1946]" gridcol="1" gridrow="14" pageId="2" pageNumber="3">1.34 ± 0.11</td>
|
||
<td id="76E55D87FFFB004910E8F8013A22F803" box="[469,568,1931,1946]" gridcol="2" gridrow="14" pageId="2" pageNumber="3">4.17 ± 0.01</td>
|
||
<td id="76E55D87FFFB004913BFF8013AEAF803" box="[642,752,1931,1946]" gridcol="3" gridrow="14" pageId="2" pageNumber="3">88.40 ± 1.43</td>
|
||
<td id="76E55D87FFFB00491204F8013BBCF803" box="[825,934,1931,1946]" gridcol="4" gridrow="14" pageId="2" pageNumber="3">3.38 ± 0.14</td>
|
||
<td id="76E55D87FFFB004912D2F8013C47F803" box="[1007,1117,1931,1946]" gridcol="5" gridrow="14" pageId="2" pageNumber="3">2.36 ± 0.34</td>
|
||
<td id="76E55D87FFFB0049159BF8013D1AF803" box="[1190,1280,1931,1946]" gridcol="6" gridrow="14" pageId="2" pageNumber="3">0.35 ± 0.01</td>
|
||
<td id="76E55D87FFFB00491474F8013DDAF803" box="[1353,1472,1931,1946]" gridcol="7" gridrow="14" pageId="2" pageNumber="3">18.73 ± 0.13</td>
|
||
</tr>
|
||
<tr id="353434FBFFFB00491149F8283DDAF828" box="[116,1472,1954,1969]" gridrow="15" pageId="2" pageNumber="3">
|
||
<th id="76E55D87FFFB00491149F82838C5F828" box="[116,223,1954,1969]" gridcol="0" gridrow="15" pageId="2" pageNumber="3">F482I/S491A</th>
|
||
<td id="76E55D87FFFB00491015F8283996F828" box="[296,396,1954,1969]" gridcol="1" gridrow="15" pageId="2" pageNumber="3">0.63 ± 0.02</td>
|
||
<td id="76E55D87FFFB004910E8F8283A22F828" box="[469,568,1954,1969]" gridcol="2" gridrow="15" pageId="2" pageNumber="3">2.72 ± 0.00</td>
|
||
<td id="76E55D87FFFB004913BFF8283AEAF828" box="[642,752,1954,1969]" gridcol="3" gridrow="15" pageId="2" pageNumber="3">92.88 ± 0.45</td>
|
||
<td id="76E55D87FFFB00491204F8283BBCF828" box="[825,934,1954,1969]" gridcol="4" gridrow="15" pageId="2" pageNumber="3">2.40 ± 0.09</td>
|
||
<td id="76E55D87FFFB004912D2F8283C47F828" box="[1007,1117,1954,1969]" gridcol="5" gridrow="15" pageId="2" pageNumber="3">1.19 ± 0.08</td>
|
||
<td id="76E55D87FFFB0049159BF8283D1AF828" box="[1190,1280,1954,1969]" gridcol="6" gridrow="15" pageId="2" pageNumber="3">0.18 ± 0.00</td>
|
||
<td id="76E55D87FFFB00491474F8283DDAF828" box="[1353,1472,1954,1969]" gridcol="7" gridrow="15" pageId="2" pageNumber="3">28.74 ± 5.73</td>
|
||
</tr>
|
||
</table>
|
||
</paragraph>
|
||
<caption id="DF7B6631FFFAFFB51159FB393AC8FB6E" ID-DOI="http://doi.org/10.5281/zenodo.8291070" ID-Zenodo-Dep="8291070" httpUri="https://zenodo.org/record/8291070/files/figure.png" pageId="3" pageNumber="4" startId="3.[100,130,1203,1220]" targetBox="[188,1399,149,1175]" targetPageId="3" targetType="figure">
|
||
<paragraph id="8BBB36B9FFFAFFB51159FB393AC8FB6E" blockId="3.[100,1487,1203,1272]" pageId="3" pageNumber="4">
|
||
<emphasis id="B970EAABFFFAFFB51159FB393885FB5D" bold="true" box="[100,159,1203,1220]" pageId="3" pageNumber="4">Fig. 2.</emphasis>
|
||
Converting pinene synthase to sabinene synthase by mutations on 482 position. Chromatograms in
|
||
<emphasis id="B970EAABFFFAFFB5153AFB393C08FB5A" bold="true" box="[1031,1042,1203,1219]" pageId="3" pageNumber="4">a</emphasis>
|
||
,
|
||
<emphasis id="B970EAABFFFAFFB51523FB393C33FB5A" bold="true" box="[1054,1065,1203,1219]" pageId="3" pageNumber="4">b</emphasis>
|
||
,
|
||
<emphasis id="B970EAABFFFAFFB5150AFB393C5BFB5A" bold="true" box="[1079,1089,1203,1219]" pageId="3" pageNumber="4">c</emphasis>
|
||
,
|
||
<emphasis id="B970EAABFFFAFFB51570FB393C43FB5A" bold="true" box="[1101,1113,1203,1219]" pageId="3" pageNumber="4">d</emphasis>
|
||
and
|
||
<emphasis id="B970EAABFFFAFFB515BBFB393C8AFB5A" bold="true" box="[1158,1168,1203,1219]" pageId="3" pageNumber="4">e</emphasis>
|
||
show the GC-MS analysis of terpene products for WT, F482A, F482I, F482V and F482T, respectively. The x-axis is the retention time and the y-axis is the relative abundance of each species. The numbers in each peak correspond to α- pinene (
|
||
<emphasis id="B970EAABFFFAFFB51097FB6C39AFFB6E" bold="true" box="[426,437,1254,1271]" pageId="3" pageNumber="4">1</emphasis>
|
||
), sabinene (
|
||
<emphasis id="B970EAABFFFAFFB5131DFB6C3A31FB6E" bold="true" box="[544,555,1254,1271]" pageId="3" pageNumber="4">2</emphasis>
|
||
) and limonene (
|
||
<emphasis id="B970EAABFFFAFFB51387FB6C3ADFFB6E" bold="true" box="[698,709,1254,1271]" pageId="3" pageNumber="4">3</emphasis>
|
||
).
|
||
</paragraph>
|
||
</caption>
|
||
<paragraph id="8BBB36B9FFFAFFB511B9F98A3A26F907" blockId="3.[100,771,1312,1973]" pageId="3" pageNumber="4">
|
||
We asked whether the combination of
|
||
<collectionCode id="ED15AE7CFFFAFFB510CFFA7539E4F98B" box="[498,510,1535,1554]" country="USA" lsid="urn:lsid:biocol.org:col:15707" name="Field Museum of Natural History, Botany Department" pageId="3" pageNumber="4" type="Herbarium">F</collectionCode>
|
||
482
|
||
<collectionCode id="ED15AE7CFFFAFFB5131DFA753A2AF98B" box="[544,560,1535,1554]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
|
||
and
|
||
<collectionCode id="ED15AE7CFFFAFFB5135DFA753A71F98B" box="[608,619,1535,1554]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="3" pageNumber="4" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFAFFB513B0FA753A84F98B" box="[653,670,1535,1554]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
|
||
mutations could affect the rigidity of the protein. After a 2 ns of molecular dynamics simulation and data analysis using MDLovoFit, we found the rigidity of
|
||
<collectionCode id="ED15AE7CFFFAFFB511F4F9D938CCF9FF" box="[201,214,1619,1638]" country="USA" lsid="urn:lsid:biocol.org:col:15707" name="Field Museum of Natural History, Botany Department" pageId="3" pageNumber="4" type="Herbarium">F</collectionCode>
|
||
482
|
||
<collectionCode id="ED15AE7CFFFAFFB511C4F9D93910F9FF" box="[249,266,1619,1638]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
|
||
/
|
||
<collectionCode id="ED15AE7CFFFAFFB5102CF9D93906F9FF" box="[273,284,1619,1638]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="3" pageNumber="4" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFAFFB51003F9D93955F9FF" box="[318,335,1619,1638]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
|
||
is similar to that of
|
||
<collectionCode id="ED15AE7CFFFAFFB5132DF9D93A01F9FF" box="[528,539,1619,1638]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="3" pageNumber="4" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFAFFB51301F9D93A57F9FF" box="[572,589,1619,1638]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
|
||
(STable 3). Apparently, the decrease of activity by the combination of
|
||
<collectionCode id="ED15AE7CFFFAFFB5136CF9E53A47F91B" box="[593,605,1647,1666]" country="USA" lsid="urn:lsid:biocol.org:col:15707" name="Field Museum of Natural History, Botany Department" pageId="3" pageNumber="4" type="Herbarium">F</collectionCode>
|
||
482
|
||
<collectionCode id="ED15AE7CFFFAFFB51342F9E53A95F91B" box="[639,655,1647,1666]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
|
||
with
|
||
<collectionCode id="ED15AE7CFFFAFFB513F9F9E53AD5F91B" box="[708,719,1647,1666]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="3" pageNumber="4" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFAFFB513CCF9E53B18F91B" box="[753,770,1647,1666]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
|
||
cannot be explained by the overall RMSD change.
|
||
</paragraph>
|
||
<paragraph id="8BBB36B9FFFAFFB511B9F92D38F2F8E4" blockId="3.[100,771,1312,1973]" pageId="3" pageNumber="4">
|
||
To further investigate this issue, we analyze the RMSF values based on the simulation data (SFig. 1
|
||
<collectionCode id="ED15AE7CFFFAFFB510B3F94939BBF94F" box="[398,417,1731,1750]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
|
||
–C). Compared with
|
||
<collectionCode id="ED15AE7CFFFAFFB51357F9493A6FF94F" box="[618,629,1731,1750]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="3" pageNumber="4" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFAFFB513AAF9493AB2F94F" box="[663,680,1731,1750]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
|
||
and WT,
|
||
<collectionCode id="ED15AE7CFFFAFFB51159F954386BF968" box="[100,113,1758,1777]" country="USA" lsid="urn:lsid:biocol.org:col:15707" name="Field Museum of Natural History, Botany Department" pageId="3" pageNumber="4" type="Herbarium">F</collectionCode>
|
||
482
|
||
<collectionCode id="ED15AE7CFFFAFFB511A8F95438BCF968" box="[149,166,1758,1777]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
|
||
/
|
||
<collectionCode id="ED15AE7CFFFAFFB51190F95438A2F968" box="[173,184,1758,1777]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="3" pageNumber="4" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFAFFB511E4F95438F0F968" box="[217,234,1758,1777]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
|
||
shows an RMSF spike at a region between residues 219 and 223. In the structural model, this region forms a loop in the protein structural model (SFig. 2). However, this loop is quite far from the active site (about 40
|
||
<collectionCode id="ED15AE7CFFFAFFB511D2F8B8391BF8D3" box="[239,257,1842,1866]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">Å</collectionCode>
|
||
). Therefore, its mobility could only affect the enzyme activity indirectly. For example, it could influence the closure of the active pocket.
|
||
</paragraph>
|
||
<paragraph id="8BBB36B9FFFAFFB511B9F80C3C39FA3A" blockId="3.[100,771,1312,1973]" lastBlockId="3.[818,1487,1312,1443]" pageId="3" pageNumber="4">
|
||
We also superimposed the structural model of WT,
|
||
<collectionCode id="ED15AE7CFFFAFFB513ABF80C3ABBF800" box="[662,673,1926,1945]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="3" pageNumber="4" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFAFFB513FFF80C3AC9F800" box="[706,723,1926,1945]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
|
||
and
|
||
<collectionCode id="ED15AE7CFFFAFFB51159F828386BF82C" box="[100,113,1954,1973]" country="USA" lsid="urn:lsid:biocol.org:col:15707" name="Field Museum of Natural History, Botany Department" pageId="3" pageNumber="4" type="Herbarium">F</collectionCode>
|
||
482
|
||
<collectionCode id="ED15AE7CFFFAFFB511A8F82838BCF82C" box="[149,166,1954,1973]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
|
||
/
|
||
<collectionCode id="ED15AE7CFFFAFFB51190F82838A2F82C" box="[173,184,1954,1973]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="3" pageNumber="4" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFAFFB511E4F82838F0F82C" box="[217,234,1954,1973]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
|
||
in complex with the docked pinyl cation (SFig. 3). In the structural model, the pinyl cation is docked between the residue 491 and 482 (SFig. 3). Interestingly, in WT and
|
||
<collectionCode id="ED15AE7CFFFAFFB5159EFAB63CB4FAD6" box="[1187,1198,1340,1359]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="3" pageNumber="4" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFAFFB515ECFAB63CF8FAD6" box="[1233,1250,1340,1359]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
|
||
, the position of the pinyl cation is similar, while in
|
||
<collectionCode id="ED15AE7CFFFAFFB51517FAD23C2DFAF2" box="[1066,1079,1368,1387]" country="USA" lsid="urn:lsid:biocol.org:col:15707" name="Field Museum of Natural History, Botany Department" pageId="3" pageNumber="4" type="Herbarium">F</collectionCode>
|
||
482
|
||
<collectionCode id="ED15AE7CFFFAFFB51561FAD23C77FAF2" box="[1116,1133,1368,1387]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
|
||
/
|
||
<collectionCode id="ED15AE7CFFFAFFB51549FAD23C65FAF2" box="[1140,1151,1368,1387]" country="Sweden" lsid="urn:lsid:biocol.org:col:15668" name="Department of Botany, Swedish Museum of Natural History" pageId="3" pageNumber="4" type="Herbarium">S</collectionCode>
|
||
491
|
||
<collectionCode id="ED15AE7CFFFAFFB5159CFAD23CA8FAF2" box="[1185,1202,1368,1387]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
|
||
, the pinyl cation is shifted towards the
|
||
<collectionCode id="ED15AE7CFFFAFFB512AFFAFE3BB9FA1E" box="[914,931,1396,1415]" country="USA" lsid="urn:lsid:biocol.org:col:15406" name="Harvard University - Arnold Arboretum" pageId="3" pageNumber="4" type="Herbarium">A</collectionCode>
|
||
482. This change in the carbocation position may impair the enzyme activity (SFig. 3).
|
||
</paragraph>
|
||
</subSubSection>
|
||
</treatment>
|
||
</document> |