Changing the substrate specificity of 2-hydroxybiphenyl 3-monooxygenase from Pseudomonas azelaica HBP1 by directed evolution

A. Meyer, A. Schmid, M. Held, A.H. Westphal, M. Röthlisberger, H.P.E. Kohler, W.J.H. van Berkel, B. Witholt

Research output: Contribution to journalArticleAcademicpeer-review

67 Citations (Scopus)

Abstract

The substrate reactivity of the flavoenzyme 2-hydroxybiphenyl 3-monooxygenase (EC 1.14.13.44, HbpA) was changed by directed evolution using error-prone PCR. In situ screening of mutant libraries resulted in the identification of proteins with increased activity towards 2-tert-butylphenol and guaiacol (2-methoxyphenol). One enzyme variant contained amino acid substitutions V368A/L417F, which were inserted by two rounds of mutagenesis. The double replacement improved the efficiency of substrate hydroxylation by reducing the uncoupled oxidation of NADH. With guaiacol as substrate, the two substitutions increased Vmax from 0.22 to 0.43 units mg1 protein and decreased the K'm from 588 to 143 ?M, improving k'cat/K'm by a factor of 8.2. With 2-tert-butylphenol as the substrate, k'cat was increased more than 5-fold. Another selected enzyme variant contained amino acid substitution I244V and had a 30␑igher specific activity with 2-sec-butylphenol, guaiacol, and the "natural" substrate 2-hydroxybiphenyl. The K'm for guaiacol decreased with this mutant, but the K'm for 2-hydroxybiphenyl increased. The primary structure of HbpA shares 20.1␜equence identity with phenol 2-monooxygenase from Trichosporon cutaneum. Structure homology modeling with this three-domain enzyme suggests that Ile244 of HbpA is located in the substrate binding pocket and is involved in accommodating the phenyl substituent of the phenol. In contrast, Val368 and Leu417 are not close to the active site and would not have been obvious candidates for modification by rational design.
Original languageEnglish
Pages (from-to)5575-5582
JournalJournal of Biological Chemistry
Volume277
DOIs
Publication statusPublished - 2002

Fingerprint

Dive into the research topics of 'Changing the substrate specificity of 2-hydroxybiphenyl 3-monooxygenase from Pseudomonas azelaica HBP1 by directed evolution'. Together they form a unique fingerprint.

Cite this