Changing the substrate reactivity of 2-hydroxybiphenyl 3-monooxygenase from Pseudomonas azelaica HBP1 by directed evolution.
The substrate reactivity of the flavoenzyme 2-hydroxybiphenyl 3-monooxygenase (EC, 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 mg(-1) protein and decreased the K'(m) from 588 to 143 microm, 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 Ile(244) of HbpA is located in the substrate binding pocket and is involved in accommodating the phenyl substituent of the phenol. In contrast, Val(368) and Leu(417) are not close to the active site and would not have been obvious candidates for modification by rational design.
Meyer
Andreas
A
Institute of Biotechnology, ETHZ, Swiss Federal Institute of Technology, ETH Hönggerberg, HPT, Zürich CH-8093, Switzerland.
Schmid
Andreas
A
Held
Martin
M
Westphal
Adrie H
AH
Rothlisberger
Martina
M
Kohler
Hans-Peter E
HP
van Berkel
Willem J H
WJ
Witholt
Bernard
B
D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D014644:Genetic Variation; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010636:Phenols / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011549:Pseudomonas / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014633:Valine / Q000737:chemistry
D010636:Phenols; D006899:Mixed Function Oxygenases; C058145:2-hydroxybiphenyl 3-monooxygenase; D007930:Leucine; D014633:Valine; C035406:2-tert-butylphenol