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Cytochrome b? reductase-cytochrome b? as an active P450 redox enzyme system in Phanerochaete chrysosporium: atypical properties and in vivo evidence of electron transfer capability to CYP63A2.

ABSTRACT: Two central redox enzyme systems exist to reduce eukaryotic P450 enzymes, the P450 oxidoreductase (POR) and the cyt b? reductase-cyt b?. In fungi, limited information is available for the cyt b(5) reductase-cyt b(5) system. Here we characterized the kinetic mechanism of (cyt b?r)-cyt b? redox system from the model white-rot fungus Phanerochaete chrysosporium (Pc) and made a quantitative comparison to the POR system. We determined that Pc-cyt b?r followed a "ping-pong" mechanism and could directly reduce cytochrome c. However, unlike other cyt b? reductases, Pc-cyt b?r lacked the typical ferricyanide reduction activity, a standard for cyt b? reductases. Through co-expression in yeast, we demonstrated that the Pc-cyt b?r-cyt b? complex is capable of transferring electrons to Pc-P450 CYP63A2 for its benzo(a)pyrene monooxygenation activity and that the efficiency was comparable to POR. In fact, both redox systems supported oxidation of an estimated one-third of the added benzo(a)pyrene amount. To our knowledge, this is the first report to indicate that the cyt b?r-cyt b? complex of fungi is capable of transferring electrons to a P450 monooxygenase. Furthermore, this is the first eukaryotic quantitative comparison of the two P450 redox enzyme systems (POR and cyt b?r-cyt b?) in terms of supporting a P450 monooxygenase activity.

SUBMITTER: Syed K

PROVIDER: S-EPMC3119499 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Cytochrome b₅ reductase-cytochrome b₅ as an active P450 redox enzyme system in Phanerochaete chrysosporium: atypical properties and in vivo evidence of electron transfer capability to CYP63A2.

Syed Khajamohiddin K Kattamuri Chandramohan C Thompson Thomas B TB Yadav Jagjit S JS

Archives of biochemistry and biophysics 20110302 1

Two central redox enzyme systems exist to reduce eukaryotic P450 enzymes, the P450 oxidoreductase (POR) and the cyt b₅ reductase-cyt b₅. In fungi, limited information is available for the cyt b(5) reductase-cyt b(5) system. Here we characterized the kinetic mechanism of (cyt b₅r)-cyt b₅ redox system from the model white-rot fungus Phanerochaete chrysosporium (Pc) and made a quantitative comparison to the POR system. We determined that Pc-cyt b₅r followed a "ping-pong" mechanism and could directl ...[more]

PMID: 21376009

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Project description:The activity of a self-sufficient cytochrome P450 enzyme, CYP505D6, from the lignin-degrading basidiomycete Phanerochaete chrysosporium was characterized. Recombinant CYP505D6 was produced in Escherichia coli and purified. In the presence of NADPH, CYP505D6 used a series of saturated fatty alcohols with C9-18 carbon chain lengths as the substrates. Hydroxylation occurred at the ?-1 to ?-6 positions of such substrates with C9-15 carbon chain lengths, except for 1-dodecanol, which was hydroxylated at the ?-1 to ?-7 positions. Fatty acids were also substrates of CYP505D6. Based on the sequence alignment, the corresponding amino acid of Tyr51, which is located at the entrance to the active-site pocket in CYP102A1, was Val51 in CYP505D6. To understand the diverse hydroxylation mechanism, wild-type CYP505D6 and its V51Y variant and wild-type CYP102A1 and its Y51V variant were generated, and the products of their reaction with dodecanoic acid were analyzed. Compared with wild-type CYP505D6, its V51Y variant generated few products hydroxylated at the ?-4 to ?-6 positions. The products generated by wild-type CYP102A1 were hydroxylated at the ?-1 to ?-4 positions, whereas its Y51V variant generated ?-1 to ?-7 hydroxydodecanoic acids. These observations indicated that Val51 plays an important role in determining the regiospecificity of fatty acid hydroxylation, at least that at the ?-4 to ?-6 positions. Aromatic compounds, such as naphthalene and 1-naphthol, were also hydroxylated by CYP505D6. These findings highlight a unique broad substrate spectrum of CYP505D6, rendering it an attractive candidate enzyme for the biotechnological industry.IMPORTANCE Phanerochaete chrysosporium is a white-rot fungus whose metabolism of lignin, aromatic pollutants, and lipids has been most extensively studied. This fungus harbors 154 cytochrome P450-encoding genes in the genome. As evidenced in this study, P. chrysosporium CYP505D6, a fused protein of P450 and its reductase, hydroxylates fatty alcohols (C9-15) and fatty acids (C9-15) at the ?-1 to ?-7 or ?-1 to ?-6 positions, respectively. Naphthalene and 1-naphthol were also hydroxylated, indicating that the substrate specificity of CYP505D6 is broader than those of the known fused proteins CYP102A1 and CYP505A1. The substrate versatility of CYP505D6 makes this enzyme an attractive candidate for biotechnological applications.

Dataset Information

Cytochrome b? reductase-cytochrome b? as an active P450 redox enzyme system in Phanerochaete chrysosporium: atypical properties and in vivo evidence of electron transfer capability to CYP63A2.

Publications

Cytochrome b₅ reductase-cytochrome b₅ as an active P450 redox enzyme system in Phanerochaete chrysosporium: atypical properties and in vivo evidence of electron transfer capability to CYP63A2.

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