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Protein Engineering of P450BM3 by Rational Redesign

By Wei-Cheng Huang

Abstract

The potential of flavocytochrome P450BM3 (CYP102A1) from Bacillus megaterium for\ud industrial chemical transformation and biotechnological application is widely acknowledged. The crystal structures of P450BM3 with fatty acid substrates bound present non-productive modes of binding of substrate with their carbons distant from the iron and the ω-terminal end in a\ud hydrophobic pocket at one side of the active site. Comparison between substrate-free and\ud substrate-bound structures of P450BM3 revealed two pockets (A-arm and B-arm) in the\ud substrate binding channel. In this thesis, A82(I/F/W) mutants in which the ‘B-arm’ pocket is filled by large hydrophobic side chains at position 82 were constructed and characterised. The A82F and A82W mutants have greater affinities for substrates (~ 800-fold) as well as being more effective catalysts of indole hydroxylation than the wild-type enzyme. The crystal structure of\ud the haem domain of the A82F mutant with bound palmitate showed different substrate binding position, in which the substrate is closer to the haem iron than wild-type enzyme. On this basis, a second series of mutants with substitutions at position 438 as well as 82, in which the ‘A-arm’ pocket is modulated by large hydrophobic side chains, were constructed and characterised. The hydroxylation of 11-methyllaurate by wild-type was found to yield traces of the ω-hydroxylated product, which is the first observation of ω-hydroxylase activity of wild-type P450BM3 to date. The mutants with both ‘B-arm’ pocket and ‘A-arm’ pocket filled with larger hydrophobic residues (A82F-T438(V/I/L/F) mutants) demonstrated 2- to 3-fold increases in the formation of\ud ω-hydroxyl-11-methyllaurate. Notably, the A82F-T438L and A82F-T438F mutants also\ud presented a marked enhancement of stereo-selectivity for styrene epoxidation to generate R-styrene oxide (~ 30-fold), suggesting that not only that these mutants of P450BM3 will be valuable catalysts for synthetically useful hydroxylation reactions but also that structure-based rational redesign will be one of the most efficient tools to generate novel biocatalysts

Publisher: University of Leicester
Year: 2008
OAI identifier: oai:lra.le.ac.uk:2381/8457

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