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Biochemical characterization and selective inhibition of β‐carotene cis–trans isomerase D27 and carotenoid cleavage dioxygenase CCD 8 on the strigolactone biosynthetic pathway
Author(s) -
Harrison Peter J.,
Newgas Sophie A.,
Descombes Flora,
Shepherd Sarah A.,
Thompson Andrew J.,
Bugg Timothy D. H.
Publication year - 2015
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.13400
Subject(s) - strigolactone , dioxygenase , enzyme , hydroxamic acid , isomerase , biochemistry , chemistry , biology , stereochemistry , arabidopsis , mutant , gene
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 CCD 7 and CCD 8 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 max 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 At CCD 7 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 At CCD 8 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 CCD 8 catalytic cycle and the existence of an essential cysteine residue in the CCD 8 active site. At CCD 8 is inhibited in a time‐dependent fashion by hydroxamic acids D2, D4, D5 and D6 (> 95% inhibition at 100 μ m ) that cause a shoot branching phenotype in A. thaliana , and selective inhibition of CCD 8 is observed using hydroxamic acids D13H and D15 (82%, 71% inhibition at 10 μ m ). The enzyme inhibition data imply that the biochemical basis of the shoot branching phenotype is due to inhibition of CCD 8.