Tryptophan-47 in the Active Site of Methylophaga Sp. Strain SK1 Flavin-monooxygenase is Important for Hydride Transfer

Abstract

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. © 2013 Elsevier Inc. All rights reserved.

Department(s)

Chemistry

Comments

National Science Foundation, Grant MCB-1021384

Keywords and Phrases

Active site residue; Flavin; Flavin monooxygenases; Hydride transfer; Kinetic isotope effects

International Standard Serial Number (ISSN)

1096-0384; 0003-9861

Document Type

Article - Journal

Document Version

Citation

File Type

text

Language(s)

English

Rights

© 2024 Elsevier, All rights reserved.

Publication Date

01 Apr 2013

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