Identification of a New Class of Cytochrome P450 from a Rhodococcus sp

Author:

Roberts Gareth A.1,Grogan Gideon1,Greter Andy1,Flitsch Sabine L.1,Turner Nicholas J.1

Affiliation:

1. The Edinburgh Centre for Protein Technology, Department of Chemistry, University of Edinburgh, Edinburgh EH9 3JJ, United Kingdom

Abstract

ABSTRACT A degenerate set of PCR primers were used to clone a gene encoding a cytochrome P450 (the P450RhF gene) from Rhodococcus sp. strain NCIMB 9784 which is of unique primary structural organization. Surprisingly, analysis of the translation product revealed that the P450 is fused to a reductase domain at the C terminus which displays sequence conservation for dioxygenase reductase proteins. The reductase partner comprises flavin mononucleotide- and NADH-binding motifs and a [2Fe2S] ferredoxin-like center. The gene was engineered for heterologous expression in Escherichia coli , and conditions were found in which the enzyme was produced in a soluble form. A recombinant strain of E. coli was able to mediate the O dealkylation of 7-ethoxycoumarin in good yield, despite the absence of any recombinant redox proteins. This unprecedented finding leads us to propose that P450RhF represents the first example of a new class of cytochromes P450 in which the reducing equivalents are supplied by a novel reductase in a fused arrangement.

Publisher

American Society for Microbiology

Subject

Molecular Biology,Microbiology

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