Characterization of Cupriavidus metallidurans CYP116B1 - A thiocarbamate herbicide oxygenating P450-phthalate dioxygenase reductase fusion protein

Ashley J. Warman, Jacob W. Robinson, Dominika Luciakova, Andrew D. Lawrence, Ker R. Marshall, Martin J. Warren, Myles R. Cheesman, Stephen E.J. Rigby, Andrew W. Munro, Kirsty J. McLean

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32 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)1675-1693
Number of pages19
JournalFEBS Journal
Volume279
Issue number9
Early online date22 Feb 2012
DOIs
Publication statusPublished - 1 May 2012
Externally publishedYes

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