title: Investigating the coenzyme specificity of phenylacetone monooxygenase from Thermobifida fusca.
abstract: Type I Baeyer-Villiger monooxygenases (BVMOs) strongly prefer NADPH over NADH as an electron donor. In order to elucidate the molecular basis for this coenzyme specificity, we have performed a site-directed mutagenesis study on phenylacetone monooxygenase (PAMO) from Thermobifida fusca. Using sequence alignments of type I BVMOs and crystal structures of PAMO and cyclohexanone monooxygenase in complex with NADP(+), we identified four residues that could interact with the 2'-phosphate moiety of NADPH in PAMO. The mutagenesis study revealed that the conserved R217 is essential for binding the adenine moiety of the nicotinamide coenzyme while it also contributes to the recognition of the 2'-phosphate moiety of NADPH. The substitution of T218 did not have a strong effect on the coenzyme specificity. The H220N and H220Q mutants exhibited a ~3-fold improvement in the catalytic efficiency with NADH while the catalytic efficiency with NADPH was hardly affected. Mutating K336 did not increase the activity of PAMO with NADH, but it had a significant and beneficial effect on the enantioselectivity of Baeyer-Villiger oxidations and sulfoxidations. In conclusion, our results indicate that the function of NADPH in catalysis cannot be easily replaced by NADH. This finding is in line with the complex catalytic mechanism and the vital role of the coenzyme in BVMOs.
authors: Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh, The Netherlands.
authors: Hanna M
authors: HM
authors: Dudek
authors: Daniel E
authors: DE
authors: Torres Pazmiño
authors: Cristina
authors: C
authors: Rodríguez
authors: Gonzalo
authors: G
authors: de Gonzalo
authors: Vicente
authors: V
authors: Gotor
authors: Marco W
authors: MW
authors: Fraaije
mesh_terms: D000096:Acetone / Q000031:analogs & derivatives* / Q000378:metabolism; D000192:Actinomycetales / Q000201:enzymology* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003067:Coenzymes / Q000378:metabolism; D015870:Gene Expression; D005818:Genetic Engineering; D007700:Kinetics; D053858:Metabolic Networks and Pathways; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017385:Sequence Homology; D013379:Substrate Specificity
chemical_list: D003067:Coenzymes; D011994:Recombinant Proteins; D000096:Acetone; D009249:NADP; D006899:Mixed Function Oxygenases; C008863:1-phenyl-2-propanone