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

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

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
Volume184
Early online date24 Apr 2018
DOIs
Publication statusPublished - Jul 2018

Fingerprint

Floxacillin
Hydroxylation
Cytochrome P-450 Enzyme System
Metabolites
Enzymes
Protein Engineering
Synthetic Chemistry Techniques
Oxacillin
Halogens
Biosynthesis
Substrates
Molecular Dynamics Simulation
Bile Ducts
Heme
Penicillins
Computer Simulation
Ducts
Mass spectrometry
Molecular dynamics
Mass Spectrometry

Keywords

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

Cite this

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title = "A combined computational and experimental study on selective flucloxacillin hydroxylation by cytochrome P450 BM3 variants",
abstract = "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.",
keywords = "Biocatalysis, Cytochrome P450 BM3, Docking, Flucloxacillin, Molecular Dynamics simulations",
author = "Luirink, {Rosa A.} and Dekker, {Stefan J.} and Luigi Capoferri and Kuiper, {Cynthia L.} and Janssen, {Laura F.H.} and Ari, {Mehmet E.} and Vermeulen, {Nico P.E.} and Vos, {J. Chris} and Commandeur, {Jan N.M.} and Geerke, {Daan P.}",
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journal = "Journal of Inorganic Biochemistry",
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A combined computational and experimental study on selective flucloxacillin hydroxylation by cytochrome P450 BM3 variants. / Luirink, Rosa A.; Dekker, Stefan J.; Capoferri, Luigi; Kuiper, Cynthia L.; Janssen, Laura F.H.; Ari, Mehmet E.; Vermeulen, Nico P.E.; Vos, J. Chris; Commandeur, Jan N.M.; Geerke, Daan P.

In: Journal of Inorganic Biochemistry, Vol. 184, 07.2018, p. 115-122.

Research output: Contribution to JournalArticleAcademicpeer-review

TY - JOUR

T1 - A combined computational and experimental study on selective flucloxacillin hydroxylation by cytochrome P450 BM3 variants

AU - Luirink, Rosa A.

AU - Dekker, Stefan J.

AU - Capoferri, Luigi

AU - Kuiper, Cynthia L.

AU - Janssen, Laura F.H.

AU - Ari, Mehmet E.

AU - Vermeulen, Nico P.E.

AU - Vos, J. Chris

AU - Commandeur, Jan N.M.

AU - Geerke, Daan P.

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