Details

Autor(en) / Beteiligte

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

Titel

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

Ist Teil von

• Journal of inorganic biochemistry, 2018-07, Vol.184, p.115-122

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

2018

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• 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. Producing the 5′-hydroxymethyl metabolite of flucloxacillin is directly relevant to drug-safety research. In a combined computational/experimental study we identified a Cytochrome P450 BM3 mutant for regioselective production of 5′-hydroxymethyl flucloxacillin and rationalized its activity in terms of preferred substrate binding in the active pose over an alternative one. [Display omitted] •A biocatalytically active mutant producing 5′-hydroxymethyl flucloxacillin is identified.•The mutant is selective for 5′-methyl hydroxylation of flucloxacillin.•Its relatively high activity is explained by preferred binding in the active pose.