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Engineering cytochrome P450 BM3 of Bacillus megaterium for terminal oxidation of palmitic acid

Paper ID Volume ID Publish Year Pages File Format Full-Text
23191 43419 2014 10 PDF Available
Title
Engineering cytochrome P450 BM3 of Bacillus megaterium for terminal oxidation of palmitic acid
Abstract

•Directed evolution of P450 BM3 for terminal fatty acid hydroxylation.•High throughput screening based on LC-MS for simultaneous detection of fatty acid metabolites.•Strong impact of residues on regioselectivity distant of the catalytic site.

Directed evolution via iterative cycles of random and targeted mutagenesis was applied to the P450 domain of the subterminal fatty acid hydroxylase CYP102A1 of Bacillus megaterium to shift its regioselectivity towards the terminal position of palmitic acid. A powerful and versatile high throughput assay based on LC-MS allowed the simultaneous detection of primary and secondary oxidation products, which was instrumental for identifying variants with a strong preference for the terminal oxidation of palmitic acid. The best variants identified acquired up to 11 amino acid alterations. Substitutions at F87, I263, and A328, relatively close to the bound substrate based on available crystallographic information contributed significantly to the altered regioselectivity. However, non-obvious residues much more distant from the bound substrate showed surprising strong contributions to the increased selectivity for the terminal position of palmitic acid.

Keywords
Cytochrome P450; Bacillus megaterium; Directed evolution; Palmitic acid; Terminal oxidation
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Engineering cytochrome P450 BM3 of Bacillus megaterium for terminal oxidation of palmitic acid
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 184, 20 August 2014, Pages 17–26
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us