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J. Biol. Chem., Vol. 277, Issue 7, 5575-5582, February 15, 2002

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Changing the Substrate Reactivity of 2-Hydroxybiphenyl 3-Monooxygenase from Pseudomonas azelaica HBP1 by Directed Evolution^*

Andreas Meyer, Andreas Schmid, Martin Held, Adrie H. Westphal^§, Martina Röthlisberger, Hans-Peter E. Kohler^¶, Willem J. H. van Berkel^§, and Bernard Witholt

From the  Institute of Biotechnology, ETHZ, Swiss Federal Institute of Technology, ETH Hönggerberg, HPT, Zürich CH-8093, Switzerland, the ^§ Laboratory of Biochemistry, Department of Agrotechnology and Food Sciences, Wageningen University, Dreijenlaan 3, Wageningen NL-6703 HA, The Netherlands, and ^¶ Environmental Microbiology and Molecular Ecotoxicology, EAWAG, Swiss Federal Institute of Environmental Sciences and Technology, Dübendorf CH-8600, Switzerland

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 V_max from 0.22 to 0.43 units mg¹ protein and decreased the K'_m from 588 to 143 µM, 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% higher 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% sequence 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.

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