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

Tobias W B Ost, Caroline S. Miles, Jane Murdoch, York Fong Cheung, Graeme A. Reid, Stephen K. Chapman, Andrew W. Munro

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    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 <C12 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. (C) 2000 Federation of European Biochemical Societies.
    Original languageEnglish
    Pages (from-to)173-177
    Number of pages4
    JournalFEBS Letters
    Issue number2
    Publication statusPublished - 8 Dec 2000


    • Cytochrome P450
    • Rational mutagenesis
    • Substrate recognition


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