Rational re‐design of the substrate binding site of flavocytochrome P450 BM3

FEBS Letters

Cheung, York-Fong³  Munro, Andrew W³  Reid, Graeme A²  Ost, Tobias W.B¹  Chapman, Stephen K¹  Murdoch, Jane²  Miles, Caroline S² 

[1] Department of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK;Institute of Cell and Molecular Biology, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JT, UK;Department of Pure and Applied Chemistry, University of Strathclyde, The Royal College, 204 George Street, Glasgow G1 1XL, UK

关键词: Cytochrome P450;    Substrate recognition;    Rational mutagenesis;   

DOI  :  10.1016/S0014-5793(00)02267-5

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Bacillus megaterium P450 BM3 is a fatty acid hydroxylase with selectivity for long chain substrates (C₁₂–C₂₀). Binding or activity with substrates of chain length 12 has not been reported. Rational mutagenesis was used to re-design the enzyme to encourage binding of short chain fatty acids (C₄–C₁₀). We show that wild-type P450 BM3 has activity and weak affinity for substrates as short as butyrate (C₄). However, turnover/binding of short chain substrates is dramatically increased by introducing a novel substrate carboxylate binding site close to the heme. Mutant L181K shows catalytic efficiency (k _cat/K _M) increased >13-fold with butyrate, while the L75T/L181K double mutant has k _cat/K _M increased >15-fold with hexanoate and binding (K _d) improved >28-fold for butyrate. Removing the arginine 47/lysine 51 carboxylate binding motif at the mouth of the active site disfavours binding of all fatty acids, indicating its importance in the initial recognition of substrates.

【 授权许可】

Unknown