title: Biochemical characterization and selective inhibition of β-carotene cis-trans isomerase D27 and carotenoid cleavage dioxygenase CCD8 on the strigolactone biosynthetic pathway.
abstract: The 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 λmax 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.
authors.affiliation: Department of Chemistry, University of Warwick, Coventry, UK.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Harrison
authors.affiliation: Department of Chemistry, University of Warwick, Coventry, UK.
authors.forename: Sophie A
authors.initials: SA
authors.lastname: Newgas
authors.affiliation: Department of Chemistry, University of Warwick, Coventry, UK.
authors.forename: Flora
authors.initials: F
authors.lastname: Descombes
authors.affiliation: Department of Chemistry, University of Warwick, Coventry, UK.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Shepherd
authors.affiliation: Cranfield Soil and Agrifood Institute, Cranfield University, Cranfield, UK.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Thompson
authors.affiliation: Department of Chemistry, University of Warwick, Coventry, UK.
authors.forename: Timothy D H
authors.initials: TD
authors.lastname: Bugg
mesh_terms: D000085:Acetates / Q000737:chemistry / Q000494:pharmacology; D017360:Arabidopsis / Q000201:enzymology; D029681:Arabidopsis Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D002338:Carotenoids / Q000737:chemistry / Q000378:metabolism; D049308:Dioxygenases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D006877:Hydroxamic Acids / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D015394:Molecular Structure; D012275:Oryza / Q000201:enzymology; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010937:Plant Growth Regulators / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D010940:Plant Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018030:Silver Compounds / Q000737:chemistry / Q000494:pharmacology; D013237:Stereoisomerism; D013379:Substrate Specificity; D019207:beta Carotene / Q000737:chemistry / Q000378:metabolism; D019745:cis-trans-Isomerases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D000085:Acetates; D029681:Arabidopsis Proteins; D004791:Enzyme Inhibitors; D006877:Hydroxamic Acids; D010446:Peptide Fragments; D010937:Plant Growth Regulators; D010940:Plant Proteins; D011994:Recombinant Proteins; D018030:Silver Compounds; C427759:beta-apo-10'-carotenal; D019207:beta Carotene; C103100:silver acetate; D002338:Carotenoids; C494441:CCD7 protein, Arabidopsis; C494442:CCD8 protein, Arabidopsis; D049308:Dioxygenases; D019745:cis-trans-Isomerases
keywords: CCD7; CCD8; D27; carotenoid cleavage dioxygenase; enzyme inhibition; hydroxamic acid; strigolactone biosynthesis