Rational evolution of a medium chain-specific cytochrome P-450 BM-3 variant

The single mutant F87A of cytochrome P-450 BM-3 from Bacillus megaterium, w as engineered by rational evolution to achieve improved hydroxylation activ ity for medium chain length substrates (C8-C10). Rational evolution combine s rational design and directed evolution to overcome the drawbacks of these methods when applied individually. Based on the X-ray structure of the enz yme, eight mutation sites (P25, V26, R47, Y51, S72, A74, L188, and M354) we re identified by modeling. Sublibraries created by site-specific randomizat ion mutagenesis of each single site were screened using a spectroscopic ass ay based on omega -p-nitrophenoxycarboxylic acids (pNCA). The mutants showi ng activity for shorter chain length substrates were combined, and these co mbi-libraries were screened again for mutants with even better catalytic pr operties. Using this approach, a P-450 BM-3 variant with five mutations (V2 6T, R47F, A74G, L188K, and F87A) that efficiently hydrolyzes 8-pNCA was obt ained. The catalytic efficiency of this mutant towards omega -p-nitrophenox ydecanoic acid (10-pNCA) and omega -p-nitrophenoxydodecanoic acid (12-pNCA) is comparable to that of the wild-type P-450 BM-3. (C) 2001 Elsevier Scien ce B.V. All rights reserved.