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

Warman, Ashley J., Robinson, Jacob W., Luciakova, Dominika, Lawrence, Andrew D., Marshall, Ker R., Warren, Martin J. ORCID: https://orcid.org/0000-0002-6028-6456, Cheesman, Myles R., Rigby, Stephen E.J., Munro, Andrew W. and McLean, Kirsty J. (2012) Characterization of Cupriavidus metallidurans CYP116B1 - A thiocarbamate herbicide oxygenating P450-phthalate dioxygenase reductase fusion protein. FEBS Journal, 279 (9). pp. 1675-1693. ISSN 1742-464X

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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.

Item Type: Article
Uncontrolled Keywords: cyp116b1 cytochrome p450 (116b1) from cupriavidus metallidurans,fusion enzyme,phthalate dioxygenase reductase,potentiometry,biochemistry,molecular biology,cell biology ,/dk/atira/pure/subjectarea/asjc/1300/1303
Faculty \ School: Faculty of Science
Faculty of Science > School of Chemistry
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Depositing User: LivePure Connector
Date Deposited: 20 Sep 2022 15:30
Last Modified: 21 Sep 2022 00:23
URI: https://ueaeprints.uea.ac.uk/id/eprint/88512
DOI: 10.1111/j.1742-4658.2012.08543.x

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