171 lines
16 KiB
XML
171 lines
16 KiB
XML
<document id="780833786816599391205974263C1DFA" ID-CLB-Dataset="55329" ID-DOI="10.1016/j.phytochem.2021.112710" ID-GBIF-Dataset="50e23bf5-4200-410f-8665-8ebf3320dae8" ID-ISSN="1873-3700" ID-Zenodo-Dep="8259095" IM.bibliography_approvedBy="diego" IM.illustrations_approvedBy="diego" IM.materialsCitations_approvedBy="diego" IM.metadata_approvedBy="felipe" IM.tables_approvedBy="diego" IM.taxonomicNames_approvedBy="diego" IM.treatments_approvedBy="diego" checkinTime="1692307719834" checkinUser="felipe" docAuthor="Tropper, Marina, H, Stephanie, ohn, Wolf, Laura-Sophie, Fritsch, Julia, Kastner-Detter, Nina, Rieck, Christoph, Munkert, Jennifer, Meitinger, Nadine, Lanig, Harald & Kreis, Wolfgang" docDate="2021" docId="3018404A9E05FFB9806DFA264710EFF1" docLanguage="en" docName="Phytochemistry.187.112710.pdf" docOrigin="Phytochemistry (112710) 187" docSource="http://dx.doi.org/10.1016/j.phytochem.2021.112710" docStyle="DocumentStyle:F36D69FC8B198FBE91029DF9C24697D3.5:Phytochemistry.2020-.journal_article" docStyleId="F36D69FC8B198FBE91029DF9C24697D3" docStyleName="Phytochemistry.2020-.journal_article" docStyleVersion="5" docTitle="Digitalis lanata subsp. leaves L." docType="treatment" docVersion="4" lastPageNumber="8" masterDocId="CC2138329E02FFBE8009FFF64229EC69" masterDocTitle="21 - Hydroxypregnane 21 - O-malonylation, a crucial step in cardenolide biosynthesis, can be achieved by substrate-promiscuous BAHD-type phenolic glucoside malonyltransferases from Arabidopsis thaliana and homolog proteins from Digitalis lanata" masterLastPageNumber="10" masterPageNumber="1" pageNumber="8" updateTime="1692639180650" updateUser="ExternalLinkService">
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<mods:title id="957D916186C671089F9204A77D76D371">21 - Hydroxypregnane 21 - O-malonylation, a crucial step in cardenolide biosynthesis, can be achieved by substrate-promiscuous BAHD-type phenolic glucoside malonyltransferases from Arabidopsis thaliana and homolog proteins from Digitalis lanata</mods:title>
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<mods:namePart id="835901B45E7FAF7AF06DB79D01F7146D">Tropper, Marina</mods:namePart>
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<mods:affiliation id="58D41F774F8DECB46DFB267559E65E39">* & Lehrstuhl für Pharmazeutische Biologie, Department Biologie, Friedrich-Alexander-Universitat ¨ Erlangen-Nürnberg, Staudtstrasse 5, 91058, Erlangen, Germany</mods:affiliation>
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<mods:namePart id="520C350CCCD74943DF15CF3D75754BAA">H, Stephanie</mods:namePart>
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<mods:affiliation id="907D50FFC37FF021F56326004EFCD8D8">* & Lehrstuhl für Pharmazeutische Biologie, Department Biologie, Friedrich-Alexander-Universitat ¨ Erlangen-Nürnberg, Staudtstrasse 5, 91058, Erlangen, Germany</mods:affiliation>
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<mods:namePart id="4FB30033838E70C2F655B9DFC10B6ACB">Fritsch, Julia</mods:namePart>
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4.6. Protein purification from fresh young
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<taxonomicName id="7FB18ADF9E05FFB981E1FA264046E98A" authorityName="L." box="[488,623,1488,1507]" class="Magnoliopsida" family="Plantaginaceae" genus="Digitalis" kingdom="Plantae" order="Lamiales" pageId="7" pageNumber="8" phylum="Tracheophyta" rank="subSpecies" species="lanata" subSpecies="leaves">Digitalis lanata</taxonomicName>
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leaves
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<paragraph id="B80EF15C9E05FFB9808DF9FE4052EACE" blockId="7.[100,770,1544,1984]" pageId="7" pageNumber="8">
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Crude extracts were prepared from fresh young leaves of
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<taxonomicName id="7FB18ADF9E05FFB982A4F9FE4128EA72" box="[685,769,1544,1563]" class="Magnoliopsida" family="Plantaginaceae" genus="Digitalis" kingdom="Plantae" order="Lamiales" pageId="7" pageNumber="8" phylum="Tracheophyta" rank="species" species="lanata">
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<emphasis id="8AC52D4E9E05FFB982A4F9FE4128EA72" bold="true" box="[685,769,1544,1563]" italics="true" pageId="7" pageNumber="8">D. lanata</emphasis>
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</taxonomicName>
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homogenized in an extraction buffer of 50 mM sodium phosphate, 30 mM β- mercaptoethanol, 20% polyvinylpolypyrrolidone (w/w), pH 7.5 at a ratio of 1:4 (w/V). The extract was stirred for 30 min and subsequently filtered through 4 layers of Miracloth (Merck KGaA, Darmstadt,
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<collectingCountry id="C0A6B1CC9E05FFB9806DF9624295EACE" box="[100,188,1684,1703]" name="Germany" pageId="7" pageNumber="8">Germany</collectingCountry>
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) before centrifugation at 48,000×
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<emphasis id="8AC52D4E9E05FFB98209F9624020EACE" bold="true" box="[512,521,1684,1703]" italics="true" pageId="7" pageNumber="8">g</emphasis>
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for 20 min.
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</paragraph>
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<paragraph id="B80EF15C9E05FFB9808DF959430DEB5B" blockId="7.[100,770,1544,1984]" pageId="7" pageNumber="8">
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Ammonium sulfate was added to the crude extracts and stirred for 30 min to obtain precipitates of 0–15%, 15–40%, 40–55% and 55–100% salt saturation. After centrifugation at 48,000×
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<emphasis id="8AC52D4E9E05FFB98228F91E4003EA92" bold="true" box="[545,554,1768,1787]" italics="true" pageId="7" pageNumber="8">g</emphasis>
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for 20 min, the pellets were dissolved in phosphate buffer (pH 7.5, 50 mM, 30 mM β- mercaptoethanol).
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</paragraph>
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<paragraph id="B80EF15C9E05FFB9808DF8CD416CED7E" blockId="7.[100,770,1544,1984]" lastBlockId="7.[818,1487,142,920]" pageId="7" pageNumber="8">
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The 40–55% and 55–100% precipitates were applied to hydrophobic interaction chromatography on Phenylsepharose FF 16/10 (low sub) (GE Healthcare Bio-Sciences AB,
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<collectingCountry id="C0A6B1CC9E05FFB981AAF88543C4EBEF" box="[419,493,1907,1926]" name="Sweden" pageId="7" pageNumber="8">Sweden</collectingCountry>
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) equilibrated with 4 column volumes of buffer A (50 mM sodium phosphate, 30 mM β- mercaptoethanol, 1 M (NH
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<subScript id="2435F3199E05FFB980F6F8444321EBA9" attach="left" box="[255,264,1970,1984]" fontSize="6" pageId="7" pageNumber="8">4</subScript>
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)
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<subScript id="2435F3199E05FFB98119F8444330EBA9" attach="right" box="[272,281,1970,1984]" fontSize="6" pageId="7" pageNumber="8">2</subScript>
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SO
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<subScript id="2435F3199E05FFB9813DF8444314EBA9" attach="left" box="[308,317,1970,1984]" fontSize="6" pageId="7" pageNumber="8">4</subScript>
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, pH 7.5). Fractions of 10 ml were collected at a flow rate of 2.0 ml min
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<superScript id="4FC45C149E05FFB98419FF784630ECF5" attach="right" box="[1040,1049,142,156]" fontSize="6" pageId="7" pageNumber="8">1</superScript>
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. Unbound proteins were flushed with 7 column volumes of buffer A and bound proteins were eluted by a decreasing salinity gradient of 0–100% of buffer B (50 mM sodium phosphate, 30 mM β- mercaptoethanol) followed by 7 column volumes of 100% buffer B.
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</paragraph>
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<paragraph id="B80EF15C9E05FFB98358FEE94710EFF1" blockId="7.[818,1487,142,920]" pageId="7" pageNumber="8">
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Fractions were tested for enzyme activity and the most active fractions were pooled and applied consecutively to ion exchange chromatography on Resource Q 1, ml (GE Healthcare Bio-Sciences AB,
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<collectingCountry id="C0A6B1CC9E05FFB98576FEA147E0ED03" box="[1407,1481,343,362]" name="Sweden" pageId="7" pageNumber="8">Sweden</collectingCountry>
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) at pH 8.0 and pH 6.5 and to Resource S, 1 ml (GE Healthcare Bio-Sciences AB,
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<collectingCountry id="C0A6B1CC9E05FFB983BBFE7941D5EDCB" box="[946,1020,399,418]" name="Sweden" pageId="7" pageNumber="8">Sweden</collectingCountry>
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) at pH 5.0 for purification of the 55–100% precipitate. The precipitate containing 40–55% salt saturation was applied to Resource Q 1 ml (GE Healthcare Bio-Sciences AB,
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<collectingCountry id="C0A6B1CC9E05FFB98528FE314742EDB3" box="[1313,1387,455,474]" name="Sweden" pageId="7" pageNumber="8">Sweden</collectingCountry>
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) at pH 8.0 and pH 8.5. The buffer of the protein solutions was exchanged before each chromatographic step using Amicon Ultra Centrifugal filter devices 1000 MWCO 20 ml (Millipore Carrightwohill,
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<collectingCountry id="C0A6B1CC9E05FFB984F3FDEC4768EE44" box="[1274,1345,538,557]" name="Ireland" pageId="7" pageNumber="8">Ireland</collectingCountry>
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). The columns were equilibrated with 4 column volumes of appropriate buffer A (
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<tableCitation id="F533C4E79E05FFB98333FDA441A3EE0C" box="[826,906,594,613]" captionStart="Table 1" captionStartId="4.[100,150,150,166]" captionTargetPageId="4" captionText="Table 1 Several 21-hydroxypregnanes as well as other compounds were docked into the homology model of AtPMaT1, AtPMaT2, DlMaT1 and their binding scores calculated. The empirical binding free energy (kcal mol 1) of the substrates in the catalytic site is shown. For acronyms and structures, see Materials & Methods, and Fig.2. Empirical binding free energies where the distance between the catalytic histidine and the hydroxy group to be acylated were deemed too large are not listed ()." httpUri="http://table.plazi.org/id/ECCEA1D49E06FFBA806DFF604305ED33" pageId="7" pageNumber="8" tableUuid="ECCEA1D49E06FFBA806DFF604305ED33">Table S1</tableCitation>
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). The flow rate was 4.0 ml min
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<superScript id="4FC45C149E05FFB984BCFDBA4697EE33" attach="right" box="[1205,1214,588,602]" fontSize="6" pageId="7" pageNumber="8">1</superScript>
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and eluates were collected in fractions of 0.5 ml. The column was washed with 10 column volumes of buffer A, and bound proteins were eluted by a gradient of 0–50% 1 M NaCl over 15 column volumes followed by 8 column volumes of 100% 1 M NaCl.Prior to SEC on Superdex 75 10/300_GL (GE Healthcare Bio-Sciences AB,
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<collectingCountry id="C0A6B1CC9E05FFB983A6FD2B41D5EE99" box="[943,1020,733,752]" name="Sweden" pageId="7" pageNumber="8">Sweden</collectingCountry>
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), the protein solution was concentrated to a sample volume of 200 μl using Amicon Ultra Centrifugal filter devices 1000 MWCO 20 ml (Millipore Carrightwohill,
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<collectingCountry id="C0A6B1CC9E05FFB984C7FCE3473CEF41" box="[1230,1301,789,808]" name="Ireland" pageId="7" pageNumber="8">Ireland</collectingCountry>
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). The column was equilibrated with 4 column volumes of sodium phosphate buffer (50 mM, 30 mM β- mercaptoethanol, 0.15 M NaCl, pH 7.5). The flow rate was set to 0.4 ml min
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and fractions of 250 μl were collected. Proteins were eluted isocratically with 1.5 column volumes of buffer.
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</paragraph>
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</subSubSection>
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</treatment>
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</document> |