Elsevier

Journal of Biotechnology

Volume 88, Issue 2, 15 June 2001, Pages 167-171
Journal of Biotechnology

Short communication
A P450 BM-3 mutant hydroxylates alkanes, cycloalkanes, arenes and heteroarenes

https://doi.org/10.1016/S0168-1656(01)00249-8Get rights and content

Abstract

P450 monooxygenases from microorganisms, similar to those of eukaryotic mitochondria, display a rather narrow substrate specificity. For native P450 BM-3, no other substrates than fatty acids or an indolyl-fatty acid derivative have been reported (Li, Q.S., Schwaneberg, U., Fischer, P., Schmid, R.D., 2000. Directed evolution of the fatty-acid hydroxylase P450BM-3 into an indole-hydroxylating catalyst. Chem. Eur. J. 6 (9), 1531–1536). Engineering the substrate specificity of Bacillus megaterium cytochrome P-450 BM3: hydroxylation of alkyl trimethylammonium compounds. Biochem. J. 327, 537–544). We thus were quite surprised to observe, in the course of our investigations on the rational evolution of this enzyme towards mutants, capable of hydroxylating shorter-chain fatty acids, that a triple mutant P450 BM-3 (Phe87Val, Leu188-Gln, Ala74Gly, BM-3 mutant) could efficiently hydroxylate indole, leading to the formation of indigo and indirubin (Li, Q.S., Schwaneberg, U., Fischer, P., Schmid, R.D., 2000. Directed evolution of the fatty-acid hydroxylase P450BM-3 into an indole-hydroxylating catalyst. Chem. Eur. J. 6 (9), 1531–1536). Indole is not oxidized by the wild-type enzyme; it lacks the carboxylate group by which the proper fatty acid substrates are supposed to be bound at the active site of the native enzyme, via hydrogen bonds to the charged amino acid residues Arg47 and Tyr51. Our attempts to predict the putative binding mode of indole to P450 BM-3 or the triple mutant by molecular dynamics simulations did not provide any useful clue. Encouraged by the unexpected activity of the triple mutant towards indole, we investigated in a preliminary, but systematic manner several alkanes, alicyclic, aromatic, and heterocyclic compounds, all of which are unaffected by the native enzyme, for their potential as substrates. We here report that this triple mutant indeed is capable to hydroxylate a respectable range of other substrates, all of which bear little or no resemblance to the fatty acid substrates of the native enzyme.

Introduction

CYP102 is a self-sufficient P450 monooxygenase from Bacillus megaterium (P450 BM-3) in which the catalytic heme domain and the FAD-containing reductase domain are naturally fused (Li and Poulos, 1997). It has been functionally expressed in Escherichia coli and is conveniently prepared and purified in gram quantities (Schwaneberg et al., 1999). Its known function is subterminal, i.e. (ωn) hydroxylation of saturated and epoxidation of unsaturated medium- and long-chain fatty acids (Boddupalli et al., 1990, Guengerich, 1991) and its specific activity in this reaction is about 1000-fold higher than eukaryotic P450 monooxygenases of a similar specificity (e.g. CYP52A3 and CYP52A4 from Candida maltosa, Scheller et al., 1996). Moreover, B. megaterium P450 BM-3 is water-soluble and thus, other than most other members of the P450 family which are multi-protein complexes attached to membranes, can be incorporated in a bioreactor using a zinc-mediator complex instead of expensive NADPH as an external electron donor (Schwaneberg et al., 2000). As the X-ray structure of P450 BM-3 has been resolved at 2.0 Å resolution (Ravichandran et al., 1993), a number of reports have appeared on the putative substrate binding site of this enzyme and have been corroborated by site-directed mutagenesis.

Section snippets

Enzyme

The P450 BM-3 (Phe78Val, Leu188Gln, Ala74Gly) mutant was obtained as described before (Li et al., 2000).

NADPH-assay

For the transformations, a 60 mM substrate solution in acetone (50 μl), 50 mM KxPO4 buffer (pH 7.5, 9.1 ml), and 2.9 nM (equivalent to 0.05 mg P450, as determined by the carbon-monoxide binding procedure) wild-type or BM-3 mutant enzyme (Li et al., 2000) were combined. For the data reported in Table 1, the mixture was pre-incubated for 5 min, and transformation was started by adding 5 mM

Results and discussion

The data in Table 1 show indeed that medium-chain alkanes as well as some alicyclic, aromatic, and heterocyclic compounds are good substrates for the BM-3 mutant though not for the wild-type enzyme. Monitoring NADPH consumption is a standard procedure to test for the substrate specificity of P450 monooxygenases (Boddupalli et al., 1990). Since one cannot differentiate, with this experimental setup, between NADPH consumption by productive processes or by side reactions, which leave the substrate

Acknowledgements

We gratefully acknowledge the help of Daniela Bezdan, Christa Kieß and Gisela Siebke for the GLC analyses, and of Dipl.-Ing. Joachim Trinkner for the GC/MS measurements. Financial support of BASF AG and of Fonds der Chemischen Industrie is gratefully acknowledged.

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1

Present address: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.

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