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

• Published in: Chembiochem : a European journal of chemical biology 2012-04, Vol.13 (6), p.872-878
• Main Authors: Jensen, Chantel N., Cartwright, Jared, Ward, Jonathan, Hart, Sam, Turkenburg, Johan P., Ali, Sohail T., Allen, Michael J., Grogan, Gideon
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Baeyer-Villiger monooxygenase; biotransformation; Catalysis; flavoprotein; Flavoproteins - chemistry; Flavoproteins - genetics; Flavoproteins - metabolism; Molecular Sequence Data; NAD - chemistry; NAD - genetics; NAD - metabolism; NADP - genetics; NADP - metabolism; NADPH; Niacinamide - chemistry; Niacinamide - genetics; Niacinamide - metabolism; Oxidation-Reduction; oxidoreductase; Oxygenases - chemistry; Oxygenases - genetics; Oxygenases - metabolism; Stenotrophomonas maltophilia - enzymology; Stenotrophomonas maltophilia - genetics; Substrate Specificity; Sulfides - chemistry; Sulfides - metabolism
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.201200006

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. A flavin monooxygenase from Stenotrophomonas maltophilia is unusual in its relaxed specificity for nicotinamide cofactors in the oxygenation of prochiral thioethers and a fused cyclobutanone substrate. The crystal structure of the enzyme reveals that the relaxed specificity may be due to the instance of a glutamine residue, instead of arginine, in the ribose 2′‐phosphate binding region for NADPH.