A P450BM-3 mutant hydroxylates alkanes, cycloalkanes, arenes and heteroarenes

P450 monooxygenases from microorganisms, similar to those of eukaryotic mit ochondria, display a rather narrow substrate specificity. For native P450 B M-3, no other substrates than fatty acids or an indolyl-fatty acid derivati ve 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 i ndole-hydroxylating catalyst. Chem. fur. J. 6 (9), 1531-1536). Engineering the substrate specificity of Bacillus megaterium cytochrome P-450 BM3: hydr oxylation of alkyl trimethylammonium compounds. Biochem. J. 327, 537-544). We thus were quite surprised to observe, in the course of our investigation s on the rational evolution of this enzyme towards mutants, capable of hydr oxylating shorter-chain fatty acids, that a triple mutant P450 BM-3 (Phe87V al, 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 h ydroxylase 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 t he 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 t he putative binding mode of indole to P450 BM-3 or the triple mutant by mol ecular dynamics simulations did not provide any useful clue. Encouraged by the unexpected activity of the triple mutant towards indole, we investigate d in a preliminary, but systematic manner several alkanes, alicyclic, aroma tic, 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 substr ates, all of which bear little or no resemblance to the fatty acid substrat es of the native enzyme. (C) 2001 Elsevier Science B.V. All rights reserved .