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<document id="F6BE1F798A5B1486F3FA0A310450696A" ID-DOI="10.1016/j.phytochem.2016.02.013" ID-ISSN="1873-3700" ID-Zenodo-Dep="10485001" IM.bibliography_approvedBy="felipe" IM.illustrations_approvedBy="julia" IM.materialsCitations_approvedBy="felipe" IM.metadata_approvedBy="felipe" IM.tables_approvedBy="julia" IM.taxonomicNames_approvedBy="julia" IM.treatments_approvedBy="julia" checkinTime="1704943003911" checkinUser="felipe" docAuthor="Hao, B., Caulfield, J. C., Hamilton, M. L., Pickett, J. A., Midega, C. A. O., Khan, Z. R., Wang, J. &amp; Hooper, A. M." docDate="2016" docId="E9788781A154FFE2EA5EF88CFC1EF90F" docLanguage="en" docName="Phytochemistry.125.73-87.pdf" docOrigin="Phytochemistry 125" docSource="http://dx.doi.org/10.1016/j.phytochem.2016.02.013" docStyle="DocumentStyle:9E596C34F4E94307D29315B03ACE1007.6:Phytochemistry.2014-2019.journal_article" docStyleId="9E596C34F4E94307D29315B03ACE1007" docStyleName="Phytochemistry.2014-2019.journal_article" docStyleVersion="6" docTitle="Desmodium incanum subsp. soluble" docType="treatment" docVersion="1" lastPageNumber="76" masterDocId="1541FFF9A157FFE6EA09FFB3FFCEFF80" masterDocTitle="Biosynthesis of natural and novel C-glycosylflavones utilising recombinant Oryza sativa C-glycosyltransferase (OsCGT) and Desmodium incanum root proteins" masterLastPageNumber="87" masterPageNumber="73" pageNumber="76" updateTime="1705610829511" updateUser="julia">
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<mods:title id="DAB9A139C61BE780F2CF2B238A995D76">Biosynthesis of natural and novel C-glycosylflavones utilising recombinant Oryza sativa C-glycosyltransferase (OsCGT) and Desmodium incanum root proteins</mods:title>
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<mods:affiliation id="5BB5BCDD0B860D364D8FEE051820E7A7">College of Science, Northwest A &amp; F University, Yangling, Shaanxi 712100, China &amp; Department of Biological Chemistry and Crop Protection, Rothamsted Research, Harpenden, Hertfordshire AL 5 2 JQ, UK</mods:affiliation>
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<mods:namePart id="5000F55A4A5C6C572EC312978EB7E372">Caulfield, J. C.</mods:namePart>
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<mods:namePart id="FDE832EC5840D821FFB462E0620538EE">Hamilton, M. L.</mods:namePart>
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<mods:namePart id="21C8ADAAAFB014BB3EB2A9E93498C793">Khan, Z. R.</mods:namePart>
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<emphasis id="53A5EA85A154FFE5EA5EF88CFD7AF8D3" box="[87,692,1855,1875]" italics="true" pageId="3" pageNumber="76">2.5. Synthesis of 6- and 8-C-glucosylflavones from synthetic 2-</emphasis>
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<emphasis id="53A5EA85A154FFE5EA5EF8E8FDA3F8EF" box="[87,621,1883,1903]" italics="true" pageId="3" pageNumber="76">
hydroxyflavanones by
<taxonomicName id="A6D14D14A154FFE5EB38F8E8FE6EF8EF" ID-CoL="3538J" authority="DC." box="[305,416,1883,1903]" class="Magnoliopsida" family="Fabaceae" genus="Desmodium" kingdom="Plantae" order="Fabales" pageId="3" pageNumber="76" phylum="Tracheophyta" rank="species" species="incanum">D. incanum</taxonomicName>
soluble root proteins
</emphasis>
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In our previous work demonstrating the biosynthesis of allelopathic
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-glycosylflavonoids found in the root exudates of
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<emphasis id="53A5EA85A154FFE5E896F81CFF6DF85E" italics="true" pageId="3" pageNumber="76">Desmodium spp</emphasis>
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., we demonstrated
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<collectionCode id="07C0AE52A154FFE5EB60F879FEBBF85E" box="[361,373,1994,2014]" country="Denmark" name="University of Copenhagen" pageId="3" pageNumber="76" type="Herbarium">C</collectionCode>
</emphasis>
-glucosylation in this genus requires a 2-hydroxyflavonone substrate (
<bibRefCitation id="05404B66A154FFE5EE61FA67FA8DFA67" author="Hamilton, M. L. &amp; Caulfield, J. C. &amp; Pickett, J. A. &amp; Hooper, A. M." box="[1128,1347,1492,1511]" pageId="3" pageNumber="76" pagination="5656 - 5659" refId="ref23386" refString="Hamilton, M. L., Caulfield, J. C., Pickett, J. A., Hooper, A. M., 2009. C - Glucosylflavonoid biosynthesis from 2 - hydroxynaringenin by Desmodium uncinatum (Jacq.) (Fabaceae). Tetrahedron Lett. 50, 5656 - 5659." type="journal article" year="2009">Hamilton et al., 2009</bibRefCitation>
). We therefore challenged the soluble root proteins of
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<emphasis id="53A5EA85A154FFE5EEF9FA5CFAAFF983" box="[1264,1377,1519,1539]" italics="true" pageId="3" pageNumber="76">D. incanum</emphasis>
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with the synthetic 2-hydroxyflavanones to assess whether there are significant differences in the capacity of
<taxonomicName id="A6D14D14A154FFE5EE96F995FADCF9BA" box="[1183,1298,1574,1594]" class="Magnoliopsida" family="Fabaceae" genus="Desmodium" kingdom="Plantae" order="Fabales" pageId="3" pageNumber="76" phylum="Tracheophyta" rank="species" species="incanum">
<emphasis id="53A5EA85A154FFE5EE96F995FADCF9BA" box="[1183,1298,1574,1594]" italics="true" pageId="3" pageNumber="76">D. incanum</emphasis>
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to
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<collectionCode id="07C0AE52A154FFE5EF32F995FA89F9BA" box="[1339,1351,1574,1594]" country="Denmark" name="University of Copenhagen" pageId="3" pageNumber="76" type="Herbarium">C</collectionCode>
</emphasis>
-glucosylate 2-hydroxyflavanones compared to the rice recombinant OsCGT protein (
<tableCitation id="2C53032CA154FFE5E989F9ECFC03F9F2" box="[896,973,1631,1650]" captionStart="Table 3" captionStartId="5.[87,131,1065,1079]" captionTargetBox="[110,1451,1427,1958]" captionTargetPageId="5" captionText="Table 3 2-Hydroxyflavanones (3) and C-glucosylflvanones (4), generated by OsCGT, introduced to D. incanum soluble root proteins with UDP-sugar donors to generate C-glucosylflavones (5,6) or di-C-glycosylflavones (711). *(Hao et al., 2015)." pageId="3" pageNumber="76">Table 3</tableCitation>
). Where standards were not available from the experiments with OsCGT which allowed NMR characterisation, the two products were analysed by high cone voltage ESIMS experiments to verify the
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<collectionCode id="07C0AE52A154FFE5E9FEF901FBCDF946" box="[1015,1027,1714,1734]" country="Denmark" name="University of Copenhagen" pageId="3" pageNumber="76" type="Herbarium">C</collectionCode>
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-glucosyl link to the flavonoid core through the loss of 90 and 120 amus (
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) (
<bibRefCitation id="05404B66A154FFE5EEAEF97CFA74F962" author="Cuyckens, F. &amp; Claeys, M." box="[1191,1466,1743,1762]" pageId="3" pageNumber="76" pagination="1 - 15" refId="ref23068" refString="Cuyckens, F., Claeys, M., 2004. Mass spectrometry in the structural analysis of flavonoids. J. Mass Spectrom. 39, 1 - 15." type="journal article" year="2004">Cuyckens and Claeys, 2004</bibRefCitation>
) (Supplementary data). An example of the mass spectrometric analysis is shown for incubation assays with
<emphasis id="53A5EA85A154FFE5EEB4F8B5FB19F899" bold="true" box="[1213,1239,1798,1817]" pageId="3" pageNumber="76">3h</emphasis>
(3
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,4
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<superScript id="96A49BDFA154FFE5EF00F8B7FAC3F890" attach="none" box="[1289,1293,1796,1808]" fontSize="5" pageId="3" pageNumber="76">0</superScript>
</emphasis>
-methylenedioxy) (
<figureCitation id="F9EA2A12A154FFE5E924F891FCAAF8B6" box="[813,868,1826,1846]" captionStart="Fig" captionStartId="4.[113,139,1507,1521]" captionTargetBox="[198,1407,690,1477]" captionTargetId="figure-277@4.[197,1408,689,1478]" captionTargetPageId="4" captionText="Fig. 2. (A) Ion scan for [M—H] — 459 showing two new metabolites found in (B) the LCMS ion trace. (C) Molecular ions of two new metabolites and (D) fragmentation under high cone voltage showing C-link fragments." figureDoi="http://doi.org/10.5281/zenodo.10485005" httpUri="https://zenodo.org/record/10485005/files/figure.png" pageId="3" pageNumber="76">Fig. 2</figureCitation>
) and in this case the products are fully characterised as
<emphasis id="53A5EA85A154FFE5EFAEF890FA0CF8B6" bold="true" box="[1447,1474,1827,1846]" pageId="3" pageNumber="76">5h</emphasis>
and
<emphasis id="53A5EA85A154FFE5E946F88DFCA4F8D1" bold="true" box="[847,874,1854,1873]" pageId="3" pageNumber="76">6h</emphasis>
. The differences between the two systems are small but relevant. OsCGT cannot
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<collectionCode id="07C0AE52A154FFE5E9F0F8EAFBCBF8ED" box="[1017,1029,1881,1901]" country="Denmark" name="University of Copenhagen" pageId="3" pageNumber="76" type="Herbarium">C</collectionCode>
</emphasis>
-glucosylate the sterically bulky
<emphasis id="53A5EA85A154FFE5EF45F8E9FAA9F8ED" bold="true" box="[1356,1383,1882,1901]" pageId="3" pageNumber="76">3n</emphasis>
(
<emphasis id="53A5EA85A154FFE5EF7CF8EAFAB3F8ED" box="[1397,1405,1881,1901]" italics="true" pageId="3" pageNumber="76">t</emphasis>
-butyl) and
<emphasis id="53A5EA85A154FFE5E95AF8C5FCA3F809" bold="true" box="[851,877,1910,1929]" pageId="3" pageNumber="76">3q</emphasis>
(phenyl) while
<taxonomicName id="A6D14D14A154FFE5EE1FF8C6FB49F809" box="[1046,1159,1909,1929]" class="Magnoliopsida" family="Fabaceae" genus="Desmodium" kingdom="Plantae" order="Fabales" pageId="3" pageNumber="76" phylum="Tracheophyta" rank="species" species="incanum">
<emphasis id="53A5EA85A154FFE5EE1FF8C6FB49F809" box="[1046,1159,1909,1929]" italics="true" pageId="3" pageNumber="76">D. incanum</emphasis>
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can, implying a relaxed steric constraint. However, this is minimal as
<emphasis id="53A5EA85A154FFE5EEA4F821FB06F825" bold="true" box="[1197,1224,1938,1957]" pageId="3" pageNumber="76">3p</emphasis>
(hexamethylenoxyl) still cannot be
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<collectionCode id="07C0AE52A154FFE5E987F81EFC54F841" box="[910,922,1965,1985]" country="Denmark" name="University of Copenhagen" pageId="3" pageNumber="76" type="Herbarium">C</collectionCode>
</emphasis>
-glycosylated by
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<emphasis id="53A5EA85A154FFE5EE4FF81EFB7BF841" box="[1094,1205,1965,1985]" italics="true" pageId="3" pageNumber="76">D. incanum</emphasis>
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as is the case with OsCGT. Minor differences include the acceptance by OsCGT of a brominated substrate
<emphasis id="53A5EA85A153FFE2EB57F9D3FEB9F9F3" bold="true" box="[350,375,1632,1651]" pageId="4" pageNumber="77">3v</emphasis>
although at a very low level and
<emphasis id="53A5EA85A153FFE2E8F2F9D3FCC1F9F3" bold="true" box="[763,783,1632,1651]" pageId="4" pageNumber="77">3i</emphasis>
(3
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,5
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-hydroxy) which was surprisingly not
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</emphasis>
-glycosylated by
<taxonomicName id="A6D14D14A153FFE2EA78F924FF2BF92B" box="[113,229,1687,1707]" class="Magnoliopsida" family="Fabaceae" genus="Desmodium" kingdom="Plantae" order="Fabales" pageId="4" pageNumber="77" phylum="Tracheophyta" rank="species" species="incanum">
<emphasis id="53A5EA85A153FFE2EA78F924FF2BF92B" box="[113,229,1687,1707]" italics="true" pageId="4" pageNumber="77">D. incanum</emphasis>
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proteins.
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significant difference was the ability of
<taxonomicName id="A6D14D14A153FFE2EA78F900FF2BF947" box="[113,229,1715,1735]" class="Magnoliopsida" family="Fabaceae" genus="Desmodium" kingdom="Plantae" order="Fabales" pageId="4" pageNumber="77" phylum="Tracheophyta" rank="species" species="incanum">
<emphasis id="53A5EA85A153FFE2EA78F900FF2BF947" box="[113,229,1715,1735]" italics="true" pageId="4" pageNumber="77">D. incanum</emphasis>
</taxonomicName>
proteins to turn over chlorinated substrates which would allow the potential for generating chlorinated biologically active synthons for cross-coupling. These results demonstrate that crude
<taxonomicName id="A6D14D14A153FFE2EAB8F8B4FEA3F89B" box="[177,365,1799,1819]" class="Magnoliopsida" family="Fabaceae" genus="Desmodium" kingdom="Plantae" order="Fabales" pageId="4" pageNumber="85" phylum="Tracheophyta" rank="subSpecies" species="incanum" subSpecies="soluble">
<emphasis id="53A5EA85A153FFE2EAB8F8B4FED1F89B" box="[177,287,1799,1819]" italics="true" pageId="4" pageNumber="77">D. incanum</emphasis>
soluble
</taxonomicName>
protein has a different, though very similar capacity to accept novel substrates. These differences may be due to a single protein having a less sterically hindered substrate specificity for the substrate B-ring (
<emphasis id="53A5EA85A153FFE2EBECF8E8FE31F8EE" bold="true" box="[485,511,1883,1902]" pageId="4" pageNumber="77">3n</emphasis>
and
<emphasis id="53A5EA85A153FFE2E83EF8E8FD9FF8EE" bold="true" box="[567,593,1883,1902]" pageId="4" pageNumber="77">3q</emphasis>
) along with some different electronic requirements (
<emphasis id="53A5EA85A153FFE2EBD0F8C4FE23F80A" bold="true" box="[473,493,1911,1930]" pageId="4" pageNumber="77">3i</emphasis>
,
<emphasis id="53A5EA85A153FFE2EBF2F8C4FDDBF80A" bold="true" box="[507,533,1911,1930]" pageId="4" pageNumber="77">3v</emphasis>
,
<emphasis id="53A5EA85A153FFE2E82AF8C4FD8DF80A" bold="true" box="[547,579,1911,1930]" pageId="4" pageNumber="77">3w</emphasis>
, and
<emphasis id="53A5EA85A153FFE2E888F8C4FD54F80A" bold="true" box="[641,666,1911,1930]" pageId="4" pageNumber="77">3x</emphasis>
) or it may be due to different
<emphasis id="53A5EA85A153FFE2EB3BF821FEF0F826" box="[306,318,1938,1958]" italics="true" pageId="4" pageNumber="77">
<collectionCode id="07C0AE52A153FFE2EB3BF821FEF0F826" box="[306,318,1938,1958]" country="Denmark" name="University of Copenhagen" pageId="4" pageNumber="77" type="Herbarium">C</collectionCode>
</emphasis>
-glycosyltransferases with a combined greater range of acceptable chemical space for 2-hydroxyflavanones. In any case, the plant has a greater capacity for producing novel
<emphasis id="53A5EA85A153FFE2E936F9ECFC85F9F3" box="[831,843,1631,1651]" italics="true" pageId="4" pageNumber="77">
<collectionCode id="07C0AE52A153FFE2E936F9ECFC85F9F3" box="[831,843,1631,1651]" country="Denmark" name="University of Copenhagen" pageId="4" pageNumber="77" type="Herbarium">C</collectionCode>
</emphasis>
-glucoslyflavones than those possible using the single rice recombinant OsCGT.
</paragraph>
</subSubSection>
</treatment>
</document>