188 lines
26 KiB
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188 lines
26 KiB
XML
<document id="A0D90BCDDC02EC13344A4618C928C1D6" ID-DOI="10.1016/j.phytochem.2020.112626" ID-ISSN="1873-3700" ID-Zenodo-Dep="8291734" IM.bibliography_approvedBy="felipe" IM.illustrations_approvedBy="valdenar" IM.materialsCitations_approvedBy="felipe" IM.metadata_approvedBy="felipe" IM.tables_approvedBy="valdenar" IM.taxonomicNames_approvedBy="valdenar" IM.treatments_approvedBy="valdenar" checkinTime="1693246558342" checkinUser="felipe" docAuthor="Miller, Justin C., Hollatz, Allison J. & Schuler, Mary A." docDate="2021" docId="8D608791DF671365FFCD2C4AFABDB246" docLanguage="en" docName="Phytochemistry.183.112626.pdf" docOrigin="Phytochemistry (112626) 183" docSource="http://dx.doi.org/10.1016/j.phytochem.2020.112626" docStyle="DocumentStyle:F36D69FC8B198FBE91029DF9C24697D3.5:Phytochemistry.2020-.journal_article" docStyleId="F36D69FC8B198FBE91029DF9C24697D3" docStyleName="Phytochemistry.2020-.journal_article" docStyleVersion="5" docTitle="Camptotheca SLAS" docType="treatment" docVersion="3" lastPageNumber="4" masterDocId="7159FFE9DF641366FFA92F64FFB3B679" masterDocTitle="P 450 variations bifurcate the early terpene indole alkaloid pathway in Catharanthus roseus and Camptotheca acuminata" masterLastPageNumber="13" masterPageNumber="1" pageNumber="4" updateTime="1693411865241" updateUser="valdenar">
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<mods:titleInfo id="4A32405162769E243771247C0CA02D9E">
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<mods:title id="A2B4DCD91C5E0C1D5D15DF900DDF3340">P 450 variations bifurcate the early terpene indole alkaloid pathway in Catharanthus roseus and Camptotheca acuminata</mods:title>
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<mods:name id="14F99C064E7486D0EAB69BA7C017B72B" type="personal">
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<mods:namePart id="7E85DCB83486B55B8F6FFBE60FF1A831">Miller, Justin C.</mods:namePart>
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<mods:affiliation id="702F78587E1414886890F31EB430F50D">Department of Chemistry, University of Illinois at Urbana-Champaign, 1201 W. Gregory Dr., 162 Edward R. Madigan Laboratory (ERML), Urbana, IL, 61801, USA</mods:affiliation>
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</mods:name>
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<mods:namePart id="67763870D7E5B701128FF19E9FF22DF1">Hollatz, Allison J.</mods:namePart>
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</mods:name>
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<mods:name id="704000B6420C93AB21E27347F87E73E8" type="personal">
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<mods:roleTerm id="D9F2A7AEA13D600415A708AC0237EB5A">Author</mods:roleTerm>
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</mods:role>
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<mods:namePart id="34C62553BD5730E8721CABBC128EAA0F">Schuler, Mary A.</mods:namePart>
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<mods:typeOfResource id="778984B1681B7E4527B9BC138CB81040">text</mods:typeOfResource>
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<mods:titleInfo id="F184CAC27A956044AD8B8F2CFD71E0E4">
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<mods:title id="BAB8F15575C01F59B117A31486774F89">Phytochemistry</mods:title>
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<mods:part id="04924A6BDCED7CB8F0AFFDC63F2921F4">
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<mods:date id="195975409A2509FF757F902D60AB64CF">2021</mods:date>
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<mods:title id="2126F516C0C9C0671E9698E868F20796">112626</mods:title>
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</mods:detail>
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<mods:detail id="7B5C83A2F9AFF8D154270ADAAA6AA8A5" type="pubDate">
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<mods:number id="2417C7DBE6812E4FDBF016FDB8CE8E7C">2021-03-31</mods:number>
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</mods:detail>
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<mods:detail id="69E2F2523515B7272B9454B5E3BF29D8" type="volume">
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<mods:number id="6ACD717B89E0C6AB3CACB5B852AF00A9">183</mods:number>
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<mods:location id="83F156A83CADB9C7D72585DBF1524D9E">
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<mods:url id="BE21AA7B5065511E5B40CDFC988F0E79">http://dx.doi.org/10.1016/j.phytochem.2020.112626</mods:url>
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<mods:classification id="BBF7B7070255786DBFD21E5AF873471D">journal article</mods:classification>
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<mods:identifier id="BFEF54AFFD47E36022A7EA5B6D4CAAA6" type="DOI">10.1016/j.phytochem.2020.112626</mods:identifier>
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<mods:identifier id="49FD0FF73A8263F9A34C4B1A220D61E5" type="ISSN">1873-3700</mods:identifier>
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<mods:identifier id="F5ABF83D5071653AB0C9BC8281ED3416" type="Zenodo-Dep">8291734</mods:identifier>
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</mods:mods>
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<treatment id="8D608791DF671365FFCD2C4AFABDB246" LSID="urn:lsid:plazi:treatment:8D608791DF671365FFCD2C4AFABDB246" httpUri="http://treatment.plazi.org/id/8D608791DF671365FFCD2C4AFABDB246" lastPageNumber="4" pageId="3" pageNumber="4">
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<subSubSection id="4DD3650CDF671365FFCD2C4AFDC7B538" box="[100,628,813,833]" pageId="3" pageNumber="4" type="nomenclature">
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<paragraph id="05763687DF671365FFCD2C4AFDC7B538" blockId="3.[100,628,813,833]" box="[100,628,813,833]" pageId="3" pageNumber="4">
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<heading id="5E3E81EBDF671365FFCD2C4AFDC7B538" bold="true" box="[100,628,813,833]" fontSize="36" level="1" pageId="3" pageNumber="4" reason="1">
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<emphasis id="37BDEA95DF671365FFCD2C4AFDC7B538" bold="true" box="[100,628,813,833]" italics="true" pageId="3" pageNumber="4">
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2.5. In vitro reconstitution of
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<taxonomicName id="C2C94D04DF671365FEDC2C49FDACB538" ID-CoL="8VVXT" ID-ENA="16921" authority="SLAS" authorityName="SLAS" box="[373,543,813,833]" class="Magnoliopsida" family="Nyssaceae" genus="Camptotheca" kingdom="Plantae" order="Cornales" pageId="3" pageNumber="4" phylum="Tracheophyta" rank="genus">Camptotheca SLAS</taxonomicName>
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activities
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</emphasis>
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</heading>
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</paragraph>
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</subSubSection>
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<subSubSection id="4DD3650CDF671365FF2D2C02FABDB246" pageId="3" pageNumber="4" type="description">
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<paragraph id="05763687DF671365FF2D2C02FAD6B7C7" blockId="3.[100,770,869,1140]" lastBlockId="3.[818,1488,147,1087]" pageId="3" pageNumber="4">
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For subsequent
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<emphasis id="37BDEA95DF671365FEB02C01FEEAB501" bold="true" box="[281,345,869,888]" italics="true" pageId="3" pageNumber="4">in vitro</emphasis>
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reconstitution assays, the His
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<subScript id="994D34C2DF671365FDDA2C09FDCFB502" attach="left" box="[627,636,877,891]" fontSize="6" pageId="3" pageNumber="4">6</subScript>
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-tagged
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<taxonomicName id="C2C94D04DF671365FD612C01FF6FB5ED" authority="CYP" authorityName="CYP" class="Magnoliopsida" family="Nyssaceae" genus="Camptotheca" kingdom="Plantae" order="Cornales" pageId="3" pageNumber="4" phylum="Tracheophyta" rank="genus">
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<emphasis id="37BDEA95DF671365FD612C01FF17B5ED" bold="true" italics="true" pageId="3" pageNumber="4">Camptotheca</emphasis>
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CYP
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</taxonomicName>
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72A564, CYP72A565 and CYP72A730 and
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<taxonomicName id="C2C94D04DF671365FD232CE5FF22B5C9" authority="CYP" authorityName="CYP" class="Magnoliopsida" family="Apocynaceae" genus="Catharanthus" kingdom="Plantae" order="Gentianales" pageId="3" pageNumber="4" phylum="Tracheophyta" rank="genus">
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<emphasis id="37BDEA95DF671365FD232CE5FCB1B5ED" bold="true" box="[650,770,897,916]" italics="true" pageId="3" pageNumber="4">Catharanthus</emphasis>
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CYP
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</taxonomicName>
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72A1v3 ORFs were cloned into the IPTG-inducible pCW bacterial expression vector and their full-length proteins were purified from
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<emphasis id="37BDEA95DF671365FD652CDDFCB1B5B5" bold="true" box="[716,770,953,972]" italics="true" pageId="3" pageNumber="4">E. coli</emphasis>
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as outlined in the experimental procedures. His
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<subScript id="994D34C2DF671365FDEC2CB8FDFDB593" attach="left" box="[581,590,988,1002]" fontSize="6" pageId="3" pageNumber="4">6</subScript>
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-tagged full-length membrane-bound forms of
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<taxonomicName id="C2C94D04DF671365FED02C95FD94B27D" authority="CPR" authorityName="CPR" box="[377,551,1009,1028]" class="Magnoliopsida" family="Nyssaceae" genus="Camptotheca" kingdom="Plantae" order="Cornales" pageId="3" pageNumber="4" phylum="Tracheophyta" rank="genus">
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<emphasis id="37BDEA95DF671365FED02C95FE5EB27D" bold="true" box="[377,493,1009,1028]" italics="true" pageId="3" pageNumber="4">Camptotheca</emphasis>
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CPR
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</taxonomicName>
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1 (His
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<subScript id="994D34C2DF671365FDC82C9CFDD9B27F" attach="left" box="[609,618,1016,1030]" fontSize="6" pageId="3" pageNumber="4">6</subScript>
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CPR1full) and CPR2 (His
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<subScript id="994D34C2DF671365FF6A2B70FF7FB25B" attach="left" box="[195,204,1044,1058]" fontSize="6" pageId="3" pageNumber="4">6</subScript>
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CPR2full) as well as truncated soluble forms of these electron transfer proteins (His
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<subScript id="994D34C2DF671365FE872B54FE84B247" attach="left" box="[302,311,1072,1086]" fontSize="6" pageId="3" pageNumber="4">6</subScript>
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CPR1Δ48, His
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<subScript id="994D34C2DF671365FE6D2B54FE7EB247" attach="left" box="[452,461,1072,1086]" fontSize="6" pageId="3" pageNumber="4">6</subScript>
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CPR2Δ68) were cloned into the IPTG-inducible pET28 bacterial expression vector and purified from
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<emphasis id="37BDEA95DF671365FFCD2B05FF2FB20A" bold="true" box="[100,156,1120,1140]" italics="true" pageId="3" pageNumber="4">E. coli</emphasis>
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. Reduced carbon monoxide difference spectra (
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<bibRefCitation id="61584B76DF671365FDCB2B05FCD1B6DE" author="Omura, T. & Sato, R." box="[610,866,148,1140]" pageId="3" pageNumber="4" pagination="556 - 561" refId="ref14851" refString="Omura, T., Sato, R., 1967. Isolation of cytochromes P- 450 and P- 420. Methods in Enzymology, Oxidation and Phosphorylation. Academic Press, pp. 556 - 561. https: // doi. org / 10.1016 / 0076 - 6879 (67) 10096 - 7." type="book chapter" year="1967">Omura and Sato, 1967</bibRefCitation>
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) used to quantify P450 levels indicated that all three
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<taxonomicName id="C2C94D04DF671365FAF52FF7FCE2B6BA" authority="His" authorityName="His" class="Magnoliopsida" family="Nyssaceae" genus="Camptotheca" kingdom="Plantae" order="Cornales" pageId="3" pageNumber="4" phylum="Tracheophyta" rank="genus">
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<emphasis id="37BDEA95DF671365FAF52FF7FA63B6DF" bold="true" box="[1372,1488,147,166]" italics="true" pageId="3" pageNumber="4">Camptotheca</emphasis>
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His
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</taxonomicName>
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<subScript id="994D34C2DF671365FCF92FD3FCEAB6BC" attach="left" box="[848,857,183,197]" fontSize="6" pageId="3" pageNumber="4">6</subScript>
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-tagged CYP72A proteins were mixtures of properly folded protein (peak around
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<quantity id="C2319B62DF671365FC122FAFFBBAB6A6" box="[955,1033,203,223]" metricMagnitude="-7" metricUnit="m" metricValue="4.5" pageId="3" pageNumber="4" unit="nm" value="450.0">450 nm</quantity>
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) with some free heme from misfolded protein (peak around
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<quantity id="C2319B62DF671365FC1A2F8CFC4FB682" box="[947,1020,232,251]" metricMagnitude="-7" metricUnit="m" metricValue="4.2" pageId="3" pageNumber="4" unit="nm" value="420.0">420 nm</quantity>
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) (
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<figureCitation id="9DF22A02DF671365FBB92F83FBFCB682" box="[1040,1103,231,251]" captionStart="Fig" captionStartId="6.[100,130,914,931]" captionTargetBox="[200,1386,149,884]" captionTargetId="figure-542@6.[199,1388,148,886]" captionTargetPageId="6" captionText="Fig. 7. Molecular models of Catharanthus CYP72A1 and Camptotheca CYP72A564 and CYP72A565. (A) Backbone overlays of Catharanthus CYP72A1 and Camptotheca CYP72A564 and CYP72A565 models are shown with the alpha-carbon RMSD amongst CYP72A1, CYP72A564 and CYP72A565 depicted from green (0.0 Å) to yellow (3.0 Å) to red (4.5 Å). (B) SRS regions in CYP72A proteins shown with predicted substrate contact residues (gray fill). (C) Identical versus (D) different side chain residues predicted within 4.5 Å of loganin (aqua) docked in Catharanthus CYP72A1 (blue) and loganic acid (gray) docked in Camptotheca CYP72A564 (orange). (E) Identical versus (F) different side chain residues predicted within 4.5 Å of loganin (aqua) docked in Catharanthus CYP72A1 (blue) and loganic acid (gray) docked in Camptotheca CYP72A565 (rose)." figureDoi="http://doi.org/10.5281/zenodo.8291748" httpUri="https://zenodo.org/record/8291748/files/figure.png" pageId="3" pageNumber="4">Fig. S7</figureCitation>
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). The calculated P450 concentrations for these
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<taxonomicName id="C2C94D04DF671365FCC12E67FC6FB76F" box="[872,988,259,278]" class="Magnoliopsida" family="Nyssaceae" genus="Camptotheca" kingdom="Plantae" order="Cornales" pageId="3" pageNumber="4" phylum="Tracheophyta" rank="genus">
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<emphasis id="37BDEA95DF671365FCC12E67FC6FB76F" bold="true" box="[872,988,259,278]" italics="true" pageId="3" pageNumber="4">Camptotheca</emphasis>
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</taxonomicName>
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preparations were in the micromolar range (15.9 μM for CYP72A564, 31.9 μM for CYP72A565, 3.34 μM for CYP72A730). Cytochrome
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<emphasis id="37BDEA95DF671365FC032E5FFC00B737" bold="true" box="[938,947,315,334]" italics="true" pageId="3" pageNumber="4">c</emphasis>
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reduction assays (
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<bibRefCitation id="61584B76DF671365FBCA2E5FFAF2B737" author="Guengerich, F. P. & Martin, M. V. & Sohl, C. D. & Cheng, Q." box="[1123,1345,315,334]" pageId="3" pageNumber="4" pagination="1245 - 1251" refId="ref13924" refString="Guengerich, F. P., Martin, M. V., Sohl, C. D., Cheng, Q., 2009. Measurement of cytochrome P 450 and NADPH - cytochrome P 450 reductase. Nat. Protoc. 4, 1245 - 1251. https: // doi. org / 10.1038 / nprot. 2009.121." type="journal article" year="2009">Guengerich et al., 2009</bibRefCitation>
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) used to quantify CPR activities indicated that the full-length and truncated His
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<subScript id="994D34C2DF671365FA112E3AFA72B715" attach="left" box="[1464,1473,350,364]" fontSize="6" pageId="3" pageNumber="4">6</subScript>
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--tagged CPR1 proteins efficiently reduced cytochrome
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<emphasis id="37BDEA95DF671365FAEF2E17FAFCB7FF" bold="true" box="[1350,1359,371,390]" italics="true" pageId="3" pageNumber="4">c</emphasis>
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whereas the full-length and truncated His
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<subScript id="994D34C2DF671365FBF62EF2FBDBB7DD" attach="left" box="[1119,1128,406,420]" fontSize="6" pageId="3" pageNumber="4">6</subScript>
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-tagged CPR2 proteins inefficiently reduced cytochrome
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<emphasis id="37BDEA95DF671365FC512ECFFBB2B7C7" bold="true" box="[1016,1025,427,446]" italics="true" pageId="3" pageNumber="4">c</emphasis>
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at equivalent protein levels (
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<figureCitation id="9DF22A02DF671365FABE2ECFFAEBB7C7" box="[1303,1368,427,446]" captionStart="Fig" captionStartId="7.[100,130,979,996]" captionTargetBox="[197,1390,149,950]" captionTargetId="figure-547@7.[196,1392,148,952]" captionTargetPageId="7" captionText="Fig. 8. Molecular models of Camptotheca CYP72A564, CYP72A565 and CYP72A730. (A) Backbone overlays of Camptotheca CYP72A730, CYP72A564 and CYP72A565 models are shown with the RMSD variance of CYP72A564 and CYP72A565 from the CYP72A730 backbone depicted in green (0.0 Å), yellow (3.0 Å) and red (4.5 Å). (B) SRS regions in CYP72A proteins shown with predicted substrate contact residues (gray fill). (C) Identical versus (D) different side chain residues predicted within 4.5 Å of loganic acid (gray) docked in Camptotheca CYP72A564 (orange) versus loganic acid (aqua) docked in CYP72A730 (magenta). (E) Identical versus (F) different side chain residues predicted within 4.5 Å of loganic acid (gray) docked in Camptotheca CYP72A565 (rose) versus loganic acid (aqua) docked in CYP72A730 (magenta)." figureDoi="http://doi.org/10.5281/zenodo.8291750" httpUri="https://zenodo.org/record/8291750/files/figure.png" pageId="3" pageNumber="4">Fig. S8</figureCitation>
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).
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</paragraph>
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<paragraph id="05763687DF671365FCF82EA3FC53B505" blockId="3.[818,1488,147,1087]" pageId="3" pageNumber="4">
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Reconstitution of the purified His
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<subScript id="994D34C2DF671365FB6D2EAAFB7EB7A5" attach="left" box="[1220,1229,462,476]" fontSize="6" pageId="3" pageNumber="4">6</subScript>
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-tagged CYP72A564 and CYP72A565 proteins with His
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<subScript id="994D34C2DF671365FBE32E8EFBE0B781" attach="left" box="[1098,1107,490,504]" fontSize="6" pageId="3" pageNumber="4">6</subScript>
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CPR1full and DLPC showed that both of these
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<taxonomicName id="C2C94D04DF671365FCC52E9AFC53B468" box="[876,992,510,529]" class="Magnoliopsida" family="Nyssaceae" genus="Camptotheca" kingdom="Plantae" order="Cornales" pageId="3" pageNumber="4" phylum="Tracheophyta" rank="genus">
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<emphasis id="37BDEA95DF671365FCC52E9AFC53B468" bold="true" box="[876,992,510,529]" italics="true" pageId="3" pageNumber="4">Camptotheca</emphasis>
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</taxonomicName>
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P450s metabolize loganic acid and loganin in the presence of NADPH (
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<figureCitation id="9DF22A02DF671365FC5E2D7EFBC5B457" box="[1015,1142,538,558]" captionStart="Fig" captionStartId="4.[100,130,994,1011]" captionTargetBox="[207,1380,149,965]" captionTargetId="figure-7@4.[206,1381,148,967]" captionTargetPageId="4" captionText="Fig. 4. LC-MS analyses of in vitro assays with purified His6-tagged CYP72A proteins reconstituted with His6-tagged CPR proteins. Reactions containing purified His6- tagged CYP72A protein, full-length His6-tagged Caa CPR1 protein (A, B) or full-length His6-tagged Caa CPR2 protein (C, D), and 250 μM loganic acid (A,C) or loganin (B,D), were incubated at 30◦C and analyzed by LC-MS as described in experimental procedures. Extracted ion chromatograms for loganic acid (m/z 375.1297), secologanic acid (m/z 373.1140), secoxyloganic acid (m/z 389.1089); loganin sodium salt (m/z +413.1418), secologanin sodium salt (m/z +411.1262), secoxyloganin (m/z 403.1246) are given with stacked chromatograms as marked." figureDoi="http://doi.org/10.5281/zenodo.8291742" httpUri="https://zenodo.org/record/8291742/files/figure.png" pageId="3" pageNumber="4">Fig. 4A and B</figureCitation>
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,
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<figureCitation id="9DF22A02DF671365FBD62D7EFB38B454" box="[1151,1163,538,557]" captionStart="Fig" captionStartId="4.[100,130,1832,1849]" captionTargetBox="[339,1246,1173,1804]" captionTargetId="figure-134@4.[338,1248,1171,1805]" captionTargetPageId="4" captionText="Fig. 5. Area of loganic acid, loganin and products from in vitro reconstitution assays conducted with full-length Camptotheca His6-tagged CPR1. Integrated areas from LC-MS analyses of purified His6-tagged CYP72A proteins reconstituted with full-length His6-tagged Caa CPR1 are shown for no NADPH (gray) and plus NADPH (gray slashed) reactions." figureDoi="http://doi.org/10.5281/zenodo.8291744" httpUri="https://zenodo.org/record/8291744/files/figure.png" pageId="3" pageNumber="4">5</figureCitation>
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). For both proteins, products from loganic acid include significant amounts of secologanic acid and smaller amounts of secoxyloganic acid—an overoxidation of the aldehyde to a carboxylic acid. Both proteins also produced secologanin from loganin. Similar reconstitutions of these two
|
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<taxonomicName id="C2C94D04DF671365FB002DEDFAAEB4E5" box="[1193,1309,649,668]" class="Magnoliopsida" family="Nyssaceae" genus="Camptotheca" kingdom="Plantae" order="Cornales" pageId="3" pageNumber="4" phylum="Tracheophyta" rank="genus">
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<emphasis id="37BDEA95DF671365FB002DEDFAAEB4E5" bold="true" box="[1193,1309,649,668]" italics="true" pageId="3" pageNumber="4">Camptotheca</emphasis>
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</taxonomicName>
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P450s with His
|
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<subScript id="994D34C2DF671365FA6F2DF5FA7CB4E6" attach="left" box="[1478,1487,657,671]" fontSize="6" pageId="3" pageNumber="4">6</subScript>
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CPR2full showed no detectable conversion of loganic acid or loganin (
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<figureCitation id="9DF22A02DF671365FC932DA5FC0EB4AC" box="[826,957,705,725]" captionStart="Fig" captionStartId="4.[100,130,994,1011]" captionTargetBox="[207,1380,149,965]" captionTargetId="figure-7@4.[206,1381,148,967]" captionTargetPageId="4" captionText="Fig. 4. LC-MS analyses of in vitro assays with purified His6-tagged CYP72A proteins reconstituted with His6-tagged CPR proteins. Reactions containing purified His6- tagged CYP72A protein, full-length His6-tagged Caa CPR1 protein (A, B) or full-length His6-tagged Caa CPR2 protein (C, D), and 250 μM loganic acid (A,C) or loganin (B,D), were incubated at 30◦C and analyzed by LC-MS as described in experimental procedures. Extracted ion chromatograms for loganic acid (m/z 375.1297), secologanic acid (m/z 373.1140), secoxyloganic acid (m/z 389.1089); loganin sodium salt (m/z +413.1418), secologanin sodium salt (m/z +411.1262), secoxyloganin (m/z 403.1246) are given with stacked chromatograms as marked." figureDoi="http://doi.org/10.5281/zenodo.8291742" httpUri="https://zenodo.org/record/8291742/files/figure.png" pageId="3" pageNumber="4">Fig. 4C and D</figureCitation>
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); reconstitutions with His
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<subScript id="994D34C2DF671365FB072DADFB04B4AE" attach="left" box="[1198,1207,713,727]" fontSize="6" pageId="3" pageNumber="4">6</subScript>
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CPR1Δ48 and His
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<subScript id="994D34C2DF671365FACC2DADFADDB4AE" attach="left" box="[1381,1390,713,727]" fontSize="6" pageId="3" pageNumber="4">6</subScript>
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CPR2Δ68 showed no conversion of loganin or loganic acid (data not shown). Reconstitutions of the more divergent CYP72A730 with His
|
|
<subScript id="994D34C2DF671365FAC02C65FAC1B576" attach="left" box="[1385,1394,769,783]" fontSize="6" pageId="3" pageNumber="4">6</subScript>
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CPR1full showed no conversion of loganic acid or loganin (
|
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<figureCitation id="9DF22A02DF671365FAB72C71FA1BB550" box="[1310,1448,789,809]" captionStart="Fig" captionStartId="4.[100,130,994,1011]" captionTargetBox="[207,1380,149,965]" captionTargetId="figure-7@4.[206,1381,148,967]" captionTargetPageId="4" captionText="Fig. 4. LC-MS analyses of in vitro assays with purified His6-tagged CYP72A proteins reconstituted with His6-tagged CPR proteins. Reactions containing purified His6- tagged CYP72A protein, full-length His6-tagged Caa CPR1 protein (A, B) or full-length His6-tagged Caa CPR2 protein (C, D), and 250 μM loganic acid (A,C) or loganin (B,D), were incubated at 30◦C and analyzed by LC-MS as described in experimental procedures. Extracted ion chromatograms for loganic acid (m/z 375.1297), secologanic acid (m/z 373.1140), secoxyloganic acid (m/z 389.1089); loganin sodium salt (m/z +413.1418), secologanin sodium salt (m/z +411.1262), secoxyloganin (m/z 403.1246) are given with stacked chromatograms as marked." figureDoi="http://doi.org/10.5281/zenodo.8291742" httpUri="https://zenodo.org/record/8291742/files/figure.png" pageId="3" pageNumber="4">Fig. 4A and B</figureCitation>
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,
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<figureCitation id="9DF22A02DF671365FA1C2C71FA72B551" box="[1461,1473,789,808]" captionStart="Fig" captionStartId="4.[100,130,1832,1849]" captionTargetBox="[339,1246,1173,1804]" captionTargetId="figure-134@4.[338,1248,1171,1805]" captionTargetPageId="4" captionText="Fig. 5. Area of loganic acid, loganin and products from in vitro reconstitution assays conducted with full-length Camptotheca His6-tagged CPR1. Integrated areas from LC-MS analyses of purified His6-tagged CYP72A proteins reconstituted with full-length His6-tagged Caa CPR1 are shown for no NADPH (gray) and plus NADPH (gray slashed) reactions." figureDoi="http://doi.org/10.5281/zenodo.8291744" httpUri="https://zenodo.org/record/8291744/files/figure.png" pageId="3" pageNumber="4">5</figureCitation>
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). Reconstitutions with full-length
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<emphasis id="37BDEA95DF671365FBCA2C55FB60B53D" bold="true" box="[1123,1235,817,836]" italics="true" pageId="3" pageNumber="4">Catharanthu</emphasis>
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s CYP72A1v3 showed conversion of loganin to secologanin but not conversion of loganic acid (
|
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<figureCitation id="9DF22A02DF671365FC932C0DFC0FB505" box="[826,956,873,892]" captionStart="Fig" captionStartId="4.[100,130,994,1011]" captionTargetBox="[207,1380,149,965]" captionTargetId="figure-7@4.[206,1381,148,967]" captionTargetPageId="4" captionText="Fig. 4. LC-MS analyses of in vitro assays with purified His6-tagged CYP72A proteins reconstituted with His6-tagged CPR proteins. Reactions containing purified His6- tagged CYP72A protein, full-length His6-tagged Caa CPR1 protein (A, B) or full-length His6-tagged Caa CPR2 protein (C, D), and 250 μM loganic acid (A,C) or loganin (B,D), were incubated at 30◦C and analyzed by LC-MS as described in experimental procedures. Extracted ion chromatograms for loganic acid (m/z 375.1297), secologanic acid (m/z 373.1140), secoxyloganic acid (m/z 389.1089); loganin sodium salt (m/z +413.1418), secologanin sodium salt (m/z +411.1262), secoxyloganin (m/z 403.1246) are given with stacked chromatograms as marked." figureDoi="http://doi.org/10.5281/zenodo.8291742" httpUri="https://zenodo.org/record/8291742/files/figure.png" pageId="3" pageNumber="4">Fig. 4A and B</figureCitation>
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,
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<figureCitation id="9DF22A02DF671365FC6F2C0DFC61B505" box="[966,978,873,892]" captionStart="Fig" captionStartId="4.[100,130,1832,1849]" captionTargetBox="[339,1246,1173,1804]" captionTargetId="figure-134@4.[338,1248,1171,1805]" captionTargetPageId="4" captionText="Fig. 5. Area of loganic acid, loganin and products from in vitro reconstitution assays conducted with full-length Camptotheca His6-tagged CPR1. Integrated areas from LC-MS analyses of purified His6-tagged CYP72A proteins reconstituted with full-length His6-tagged Caa CPR1 are shown for no NADPH (gray) and plus NADPH (gray slashed) reactions." figureDoi="http://doi.org/10.5281/zenodo.8291744" httpUri="https://zenodo.org/record/8291744/files/figure.png" pageId="3" pageNumber="4">5</figureCitation>
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).
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</paragraph>
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<paragraph id="05763687DF671365FCF82CE1FABDB246" blockId="3.[818,1488,147,1087]" pageId="3" pageNumber="4">
|
|
Lacking access to the 7-deoxyloganic acid needed to assess the 7-hydroxylation activity of these CYPs reported by
|
|
<bibRefCitation id="61584B76DF671365FB5F2CC5FA15B5CD" author="Yang, Y. & Li, W. & Pang, J. & Jiang, L. & Qu, X. & Pu, X. & Zhang, G. & Luo, Y." box="[1270,1446,929,948]" pageId="3" pageNumber="4" pagination="1091 - 1096" refId="ref15782" refString="Yang, Y., Li, W., Pang, J., Jiang, L., Qu, X., Pu, X., Zhang, G., Luo, Y., 2019. Bifunctional cytochrome P 450 enzymes involved in camptothecin biosynthesis. ACS Chem. Biol. 14, 1091 - 1096. https: // doi. org / 10.1021 / acschembio. 8 b 01124." type="journal article" year="2019">Yang et al. (2019)</bibRefCitation>
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, we assayed the structurally-related geniposide listed as an alternate substrate. Although we observed product formation consistent with geniposide hydroxylation for CYP72A1, CYP72A564, and CYP72A565 reconstituted with
|
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<taxonomicName id="C2C94D04DF671365FC442B74FB38B25A" authority="His" authorityName="His" box="[1005,1163,1040,1059]" class="Magnoliopsida" family="Nyssaceae" genus="Camptotheca" kingdom="Plantae" order="Cornales" pageId="3" pageNumber="4" phylum="Tracheophyta" rank="genus">
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<emphasis id="37BDEA95DF671365FC442B74FBD2B25A" bold="true" box="[1005,1121,1040,1059]" italics="true" pageId="3" pageNumber="4">Camptotheca</emphasis>
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His
|
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</taxonomicName>
|
|
<subScript id="994D34C2DF671365FB232B73FB20B25C" attach="left" box="[1162,1171,1047,1061]" fontSize="6" pageId="3" pageNumber="4">6</subScript>
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CPR1full, no such activity was observed for CYP72A730 (Supplemental Fig. S13).
|
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</paragraph>
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</subSubSection>
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</treatment>
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</document> |