title: Characterization of Cupriavidus metallidurans CYP116B1--a thiocarbamate herbicide oxygenating P450-phthalate dioxygenase reductase fusion protein.
abstract: The novel cytochrome P450/redox partner fusion enzyme CYP116B1 from Cupriavidus metallidurans was expressed in and purified from Escherichia coli. Isolated CYP116B1 exhibited a characteristic Fe(II)CO complex with Soret maximum at 449 nm. EPR and resonance Raman analyses indicated low-spin, cysteinate-coordinated ferric haem iron at both 10 K and ambient temperature, respectively, for oxidized CYP116B1. The EPR of reduced CYP116B1 demonstrated stoichiometric binding of a 2Fe-2S cluster in the reductase domain. FMN binding in the reductase domain was confirmed by flavin fluorescence studies. Steady-state reduction of cytochrome c and ferricyanide were supported by both NADPH/NADH, with NADPH used more efficiently (K(m[NADPH]) = 0.9 ± 0.5 μM and K(m[NADH]) = 399.1 ± 52.1 μM). Stopped-flow studies of NAD(P)H-dependent electron transfer to the reductase confirmed the preference for NADPH. The reduction potential of the P450 haem iron was -301 ± 7 mV, with retention of haem thiolate ligation in the ferrous enzyme. Redox potentials for the 2Fe-2S and FMN cofactors were more positive than that of the haem iron. Multi-angle laser light scattering demonstrated CYP116B1 to be monomeric. Type I (substrate-like) binding of selected unsaturated fatty acids (myristoleic, palmitoleic and arachidonic acids) was shown, but these substrates were not oxidized by CYP116B1. However, CYP116B1 catalysed hydroxylation (on propyl chains) of the herbicides S-ethyl dipropylthiocarbamate (EPTC) and S-propyl dipropylthiocarbamate (vernolate), and the subsequent N-dealkylation of vernolate. CYP116B1 thus has similar thiocarbamate-oxidizing catalytic properties to Rhodoccocus erythropolis CYP116A1, a P450 involved in the oxidative degradation of EPTC.
authors.affiliation: Department of Biochemistry, University of Leicester, Leicester, UK.
authors.forename: Ashley J
authors.initials: AJ
authors.lastname: Warman
authors.forename: Jacob W
authors.initials: JW
authors.lastname: Robinson
authors.forename: Dominika
authors.initials: D
authors.lastname: Luciakova
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Lawrence
authors.forename: Ker R
authors.initials: KR
authors.lastname: Marshall
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Warren
authors.forename: Myles R
authors.initials: MR
authors.lastname: Cheesman
authors.forename: Stephen E J
authors.initials: SE
authors.lastname: Rigby
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Munro
authors.forename: Kirsty J
authors.initials: KJ
authors.lastname: McLean
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D003001:Cloning, Molecular; D049921:Cupriavidus / Q000201:enzymology; D003486:Cyanides / Q000494:pharmacology; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006540:Herbicides / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D007834:Lasers; D009249:NADP / Q000378:metabolism; D009569:Nitric Oxide / Q000494:pharmacology; D010088:Oxidoreductases / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012240:Rhodococcus / Q000201:enzymology; D012542:Scattering, Radiation; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D013859:Thiocarbamates / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003486:Cyanides; D065607:Cytochrome P-450 Enzyme Inhibitors; D006540:Herbicides; D007093:Imidazoles; D007506:Iron-Sulfur Proteins; D011993:Recombinant Fusion Proteins; D013859:Thiocarbamates; D009569:Nitric Oxide; D009249:NADP; C029899:imidazole; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; C049135:phthalate oxygenase reductase