Browsing by Author "Harrison, Peter J."

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    Biochemical characterization and selective inhibition of β-carotene cis-trans isomerase D27 and carotenoid cleavage dioxygenase CCD8 on the strigolactone biosynthetic pathway
    (Wiley, 2015-08-31) Harrison, Peter J.; Newgas, Sophie A.; Decombes, Flora; Shepherd, Sarah A.; Thompson, Andrew J.; Bugg, Timothy D. H.
    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 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.
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    In vitro assay and inhibition of 9-cis-epoxycarotenoid dioxygenase (NCED) from Solanum lycopersicum and Zea mays
    (Elsevier, 2024-09-17) Harrison, Peter J.; Chandler, Jake; Thompson, Andrew J.; Bugg, Timothy D. H.
    The article reports methods for the expression and assay of 9-cis-epoxycarotenoid cleavage dioxygenase (NCED), an enzyme involved in the biosynthesis of phytohormone abscisic acid in plants. A method for the preparation of the unstable substrate 9′-cis-neoxanthin from fresh spinach is described. The inhibition of Solanum lycopersicum NCED by a series of aryl hydroxamic acid inhibitors is illustrated, and inhibitors D2 and D4 are assayed against NCED isozymes from Zea mays.
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    Promotion of germination using hydroxamic acid inhibitors of 9-cis-epoxycarotenoid dioxygenase
    (Frontiers Media, 2017-03-01) Awan, Sajjad Zahoor; Chandler, Jake O.; Harrison, Peter J.; Sergeant, Martin J.; Bugg, Timothy D.; Thompson, Andrew J.
    Abscisic acid (ABA) inhibits seed germination and the regulation of ABA biosynthesis has a role in maintenance of seed dormancy. The key rate-limiting step in ABA biosynthesis is catalysed by 9-cis-epoxycarotenoid dioxygenase (NCED). Two hydroxamic acid inhibitors of carotenoid cleavage dioxygenase (CCD), D4 and D7, previously found to inhibit CCD and NCED in vitro, are shown to have the novel property of decreasing mean germination time of tomato (Solanum lycopersicum L.) seeds constitutively overexpressing LeNCED1. Post-germination, D4 exhibited no negative effects on tomato seedling growth in terms of height, dry weight and fresh weight. Tobacco (Nicotiana tabacum L.) seeds containing a tetracycline-inducible LeNCED1 transgene were used to show that germination could be negatively and positively controlled through the chemical induction of gene expression and the chemical inhibition of the NCED protein: application of tetracycline increased mean germination time and delayed hypocotyl emergence in a similar manner to that observed when exogenous ABA was applied and this was reversed by D4 when NCED expression was induced at intermediate levels. D4 also improved germination in lettuce (Lactuca sativa L.) seeds under thermoinhibitory temperatures and in tomato seeds imbibed in high osmolarity solutions of polyethylene glycol. D4 reduced ABA and dihydrophaseic acid accumulation in tomato seeds overexpressing LeNCED1 and reduced ABA accumulation in wild type tomato seeds imbibed on polyethylene glycol. The evidence supports a mode of action of D4 through NCED inhibition, and this molecule provides a lead compound for the design of NCED inhibitors with greater specificity and potency.