Tryptophan-47 in the active site of Methylophaga sp. strain SK1 flavin-monooxygenase is important for hydride transfer.
Flavin-dependent monooxygenase (FMO) from Methylophaga sp. strain SK1 catalyzes the NADPH- and oxygen-dependent hydroxylation of a number of xenobiotics. Reduction of the flavin cofactor by NADPH is required for activation of molecular oxygen. The role of a conserved tryptophan at position 47 was probed by site-directed mutagenesis. FMOW47A resulted in an insoluble inactive protein; in contrast, FMOW47F was soluble and active. The spectrum of the flavin in the mutant enzyme was redshifted, indicating a change in the flavin environment. The kcat values for NADPH, trimethylamine, and methimazole, decreased 5-8-fold. Primary kinetic isotope effect values were higher, indicating that hydride transfer is more rate-limiting in the mutant enzyme. This is supported by a decrease in the rate constant for flavin reduction and in the solvent kinetic isotope effect values. Results from molecular dynamics simulations show reduced flexibility in active site residues and, in particular, the nicotinamide moiety of NADP+ in FMOW47F. This was supported by thermal denaturation experiments. Together, the data suggests that W47 plays a role in maintaining the overall protein flexibility that is required for conformational changes important in hydride transfer.
Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA.
Andre
A
Han
Reeder M
RM
Robinson
Somayesadat
S
Badieyan
Jacob
J
Ellerbrock
Pablo
P
Sobrado
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005415:Flavins / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D044147:Piscirickettsiaceae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D055550:Protein Stability; D016415:Sequence Alignment; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D005415:Flavins; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D014364:Tryptophan; D006899:Mixed Function Oxygenases