title: A flavoprotein monooxygenase that catalyses a Baeyer-Villiger reaction and thioether oxidation using NADH as the nicotinamide cofactor.
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 K(M)  and k(cat)  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, k(cat) /K(M)  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.
authors.affiliation: Department of Chemistry, University of York, Heslington, York, YO10 5DD, UK.
authors.forename: Chantel N
authors.initials: CN
authors.lastname: Jensen
authors.forename: Jared
authors.initials: J
authors.lastname: Cartwright
authors.forename: Jonathan
authors.initials: J
authors.lastname: Ward
authors.forename: Sam
authors.initials: S
authors.lastname: Hart
authors.forename: Johan P
authors.initials: JP
authors.lastname: Turkenburg
authors.forename: Sohail T
authors.initials: ST
authors.lastname: Ali
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Allen
authors.forename: Gideon
authors.initials: G
authors.lastname: Grogan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D005420:Flavoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009243:NAD / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009249:NADP / Q000235:genetics / Q000378:metabolism; D009536:Niacinamide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020615:Stenotrophomonas maltophilia / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity; D013440:Sulfides / Q000737:chemistry* / Q000378:metabolism
chemical_list: D005420:Flavoproteins; D013440:Sulfides; D009243:NAD; D009536:Niacinamide; D009249:NADP; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming)