Biochemical characterization and selective inhibition of β-carotene cis-trans isomerase D27 and carotenoid cleavage dioxygenase CCD8 on the strigolactone biosynthetic pathway

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dc.contributor.authorHarrison, Peter J.
dc.contributor.authorNewgas, Sophie A.
dc.contributor.authorDecombes, Flora
dc.contributor.authorShepherd, Sarah A.
dc.contributor.authorThompson, Andrew J.
dc.contributor.authorBugg, Timothy D. H.
dc.date.accessioned2016-10-27T18:36:16Z
dc.date.available2016-10-27T18:36:16Z
dc.date.issued2015-08-31
dc.description.abstractThe first three enzymatic steps of the strigolactone biosynthetic pathway catalysed by β-carotene cis-trans isomerase Dwarf27 (D27) from Oryza sativa and carotenoid cleavage dioxygenases CCD7 and CCD8 from Arabidopsis thaliana have been reconstituted in vitro, and kinetic assays have been developed for each enzyme, in order to develop selective enzyme inhibitors. Recombinant OsD27 shows a UV-visible λ at 422 nm and is inactivated by silver(I) acetate, consistent with the presence of an iron-sulfur cluster that is used in catalysis. OsD27 and AtCCD7 are not inhibited by hydroxamic acids that cause shoot branching in planta, but OsD27 is partially inhibited by terpene-like hydroxamic acids. The reaction catalysed by AtCCD8 is shown to be a two-step kinetic mechanism using pre-steady-state kinetic analysis. Kinetic evidence is presented for acid-base catalysis in the CCD8 catalytic cycle and the existence of an essential cysteine residue in the CCD8 active site. AtCCD8 is inhibited in a time-dependent fashion by hydroxamic acids D2, D4, D5 and D6 (> 95% inhibition at 100 μm) that cause a shoot branching phenotype in A. thaliana, and selective inhibition of CCD8 is observed using hydroxamic acids D13H and D15 (82%, 71% inhibition at 10 μm). The enzyme inhibition data imply that the biochemical basis of the shoot branching phenotype is due to inhibition of CCD8.en_UK
dc.identifier.citationHarrison, P. J., Newgas, S. A., Descombes, F., Shepherd, S. A., Thompson, A. J. and Bugg, T. D. H. (2015), Biochemical characterization and selective inhibition of β-carotene cis–trans isomerase D27 and carotenoid cleavage dioxygenase CCD8 on the strigolactone biosynthetic pathway. The FEBS Journal, Volume 282, Issue 20, 2015, pp. 3986–4000.en_UK
dc.identifier.cris1885318
dc.identifier.issn1742-464X
dc.identifier.urihttp://dx.doi.org/10.1111/febs.13400
dc.identifier.urihttp://dspace.lib.cranfield.ac.uk/handle/1826/10901
dc.language.isoenen_UK
dc.publisherWileyen_UK
dc.rightsAttribution-Non-Commercial 3.0 Unported (CC BY-NC 3.0) You are free to: Share — copy and redistribute the material in any medium or format, Adapt — remix, transform, and build upon the material. The licensor cannot revoke these freedoms as long as you follow the license terms. Under the following terms: Attribution — You must give appropriate credit, provide a link to the license, and indicate if changes were made. You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use. Information: Non-Commercial — You may not use the material for commercial purposes. No additional restrictions — You may not apply legal terms or technological measures that legally restrict others from doing anything the license permits.en_UK
dc.rightsThis is the peer reviewed version of the following article: Harrison, P. J., Newgas, S. A., Descombes, F., Shepherd, S. A., Thompson, A. J. and Bugg, T. D. H. (2015), Biochemical characterization and selective inhibition of β-carotene cis–trans isomerase D27 and carotenoid cleavage dioxygenase CCD8 on the strigolactone biosynthetic pathway. FEBS J, 282: 3986–4000., which has been published in final form athttp://dx.doi.org/10.1111/febs.13400 . This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. http://olabout.wiley.com/WileyCDA/Section/id-828039.html#submitteden_UK
dc.titleBiochemical characterization and selective inhibition of β-carotene cis-trans isomerase D27 and carotenoid cleavage dioxygenase CCD8 on the strigolactone biosynthetic pathwayen_UK
dc.typeArticleen_UK

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