Modification of the fatty acid specificity of cytochrome P450BM-3 from Bacillus megaterium by directed evolution: A validated assay

Cytochrome P450 BM-3 (CYP102) catalyzes the subterminal hydroxylation of fa tty acids with a chain length of 12-22 carbons. The paper focuses on the re gioselectivity and substrate specificity of the purified wild-type enzyme a nd five mutated variants towards caprylic, capric, and lauric acid. The enz ymes were obtained by random mutagenic fine-tuning of the mutant F87A(LARV) . F87A(LARV) was selected as the best enzyme variant in a previous study in which the single mutant F87A was subjected to rational evolution to achiev e hydroxylation activity for short chain length substrates using ap-nitroph enolate-based spectrophotometric assay. The best mutants, F87V(LAR) and F87V(LARV), show a higher catalytic activit y towards omega-(p-nitrophenoxy)decanoic acid (W-p-NCA) than F87A(LARV). In addition, they proved capable of hydroxylating omega-(p-nitrophenoxy)octan oic acid (8-p-NCA) which the wild-type enzyme is unable to do. Both variant s catalyzed hydroxylation of capric acid, which is not a substrate for the wild-type, with a conversion rate of up to 57%. The chain length specificit y of the mutants in fatty acid hydroxylation processes shows a good correla tion with their activity towards p-NCA pseudosubstrates. The p-NCA assay th erefore, allows high-throughput screening of large mutant libraries for the identification of enzyme variants with the desired catalytic activity towa rds fatty acids as the natural substrates. (C) 2001 Elsevier Science B.V. A ll rights reserved.