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Journal of Bacteriology, July 2002, p. 3898-3908, Vol. 184, No. 14
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.14.3898-3908.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Gareth A. Roberts, Gideon Grogan,,{dagger} Andy Greter, Sabine L. Flitsch, and Nicholas J. Turner*

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

Received 22 January 2002/ Accepted 26 April 2002

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.

* Corresponding author. Mailing address: The Edinburgh Centre for Protein Technology, Department of Chemistry, University of Edinburgh, The King's Buildings, West Mains Rd., Edinburgh EH9 3JJ, United Kingdom. Phone: 44-131-650-4717. Fax: 44-131-650-4743. E-mail: n.j.turner{at}ed.ac.uk.

{dagger} Present address: Department of Chemistry, The University of York, Heslington, York YO10 5DD, United Kingdom.

Journal of Bacteriology, July 2002, p. 3898-3908, Vol. 184, No. 14
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.14.3898-3908.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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