A Flavoprotein Monooxygenase that Catalyses a Baeyer-Villiger Reaction and Thioether Oxidation Using NADH as the Nicotinamide Cofactor

Jensen, Chantel N. and Cartwright, Jared and Ward, Jonathan and Hart, Sam and Turkenburg, Johan P. and Ali, Sohail T. and Allen, Michael J. and Grogan, Gideon (2012) A Flavoprotein Monooxygenase that Catalyses a Baeyer-Villiger Reaction and Thioether Oxidation Using NADH as the Nicotinamide Cofactor. ChemBioChem, 13 (6). pp. 872-878. ISSN 1439-4227

Abstract

A gene from the marine bacterium Stenotrophomonas maltophilia encodes a 38.6 kDa FAD-containing flavoprotein (Uniprot B2FLR2) named S. maltophilia flavin-containing monooxygenase (SMFMO), which catalyses the oxidation of thioethers and also the regioselective Baeyer–Villiger oxidation of the model substrate bicyclo[3.2.0]hept-2-en-6-one. The enzyme was unusual in its ability to employ either NADH or NADPH as nicotinamide cofactor. The KM and kcat values for NADH were 23.7±9.1 μM and 0.029 s−1 and 27.3±5.3 μM and 0.022 s−1 for NADPH. However, kcat/KM value for the ketone substrate in the presence of 100 μM cofactor was 17 times greater for NADH than for NADPH. SMFMO catalysed the quantitative conversion of 5 mM ketone in the presence of substoichiometric concentrations of NADH with the formate dehydrogenase cofactor recycling system, to give the 2-oxa and 3-oxa lactone products of Baeyer–Villiger reaction in a ratio of 5:1, albeit with poor enantioselectivity. The conversion with NADPH was 15 %. SMFMO also catalysed the NADH-dependent transformation of prochiral aromatic thioethers, giving in the best case, 80 % ee for the transformation of p-chlorophenyl methyl sulfide to its R enantiomer. The structure of SMFMO reveals that the relaxation in cofactor specificity appears to be accomplished by the substitution of an arginine residue, responsible for recognition of the 2′-phosphate on the NADPH ribose in related NADPH-dependent FMOs, with a glutamine residue in SMFMO. SMFMO is thus representative of a separate class of single-component, flavoprotein monooxygenases that catalyse NADH-dependent oxidations from which possible sequences and strategies for developing NADH-dependent biocatalysts for asymmetric oxygenation reactions might be identified.