A combined computational and experimental study on selective flucloxacillin hydroxylation by cytochrome P450 BM3 variants

Rosa A. Luirink, Stefan J. Dekker, Luigi Capoferri, Cynthia L. Kuiper, Laura F.H. Janssen, Mehmet E. Ari, Nico P.E. Vermeulen, J. Chris Vos, Jan N.M. Commandeur, Daan P. Geerke*

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review

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The 5′-hydroxymethyl metabolite of the penicillin based antibiotic flucloxacillin (FLX) is considered to be involved in bile duct damage occurring in a small number of patients. Because 5′-hydroxymethyl FLX is difficult to obtain by organic synthesis, biosynthesis using highly active and regioselective biocatalysts would be an alternative approach. By screening an in-house library of Cytochrome P450 (CYP) BM3 mutants, mutant M11 L437E was identified as a regioselective enzyme with relatively high activity in production of 5′-hydroxymethyl FLX as was confirmed by mass spectrometry and NMR. In contrast, incubation of M11 L437E and other mutants with oxacillin (OX, which differs from FLX by a lack of aromatic halogens) resulted in formation of two metabolites. In addition to 5′-hydroxymethyl OX we identified a product resulting from aromatic hydroxylation. In silico studies of both FLX and OX with three CYP BM3 mutants revealed substrate binding poses allowing for 5′-methyl hydroxylation, as well as binding poses with the aromatic moiety in the vicinity of the heme iron for which the corresponding product of aromatic hydroxylation was not observed for FLX. Supported by the (differences in) experimentally determined ratios of product formation for OX hydroxylation by M11 and its L437A variant and M11 L437E, Molecular Dynamics simulations suggest that the preference of mutant M11 L437E to bind FLX in its catalytically active pose over the other binding orientation contributes to its biocatalytic activity, highlighting the benefit of studying effects of active-site mutations on possible alternative enzyme-substrate binding poses in protein engineering.

Original languageEnglish
Pages (from-to)115-122
Number of pages8
JournalJournal of Inorganic Biochemistry
Early online date24 Apr 2018
Publication statusPublished - Jul 2018


This work was supported by the Netherlands Organization for Scientific Research (NWO, VIDI grant 723.012.105) and by the European Community under the Innovative Medicines Initiative (IMI) program through Grant Agreement number 115336. Appendix A

FundersFunder number
European Community115336
Netherlands Organization for Scientific Research
Nederlandse Organisatie voor Wetenschappelijk Onderzoek


    • Biocatalysis
    • Cytochrome P450 BM3
    • Docking
    • Flucloxacillin
    • Molecular Dynamics simulations


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