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.

A Comparative Linear Interaction Energy and MM/PBSA Study on SIRT1–Ligand Binding Free Energy Calculation

“…73 The MD trajectory and structure file formats are imported using the MDTraj library. 74 MD trajectories were analyzed for the following biomolecular interaction types at a 100 ps resolution similar to previous studies performed by us: 35,61,75 Hydrophobic interactions : identified as all contacts between carbon atoms within 0.4 nm having only C or H atoms as neighbors. Interacting pairs from the same source atom to multiple target atoms in the same residue, in both directions, were filtered to retain only the pair with the smallest distance.…”

A Modified Arrhenius Approach to Thermodynamically Study Regioselectivity in Cytochrome P450‐Catalyzed Substrate Conversion

“…All MD simulations (including thermal equilibration and 100 ns production simulations) were performed using identical simulation settings and force-field parameters for the protein and heme group (either in the resting or the compound Is tate) as described in ref. [74]. Atomic coordinates were written out to disk every 100 ps.…”

Characterization of kinetics of human cytochrome P450s involved in bioactivation of flucloxacillin: inhibition of CYP3A‐catalysed hydroxylation by sulfaphenazole

“…5′‐HM‐FLX was obtained biosynthetically by hydroxylation of flucloxacillin using a drug metabolizing mutant of CYP102A1 from Bacillus megaterium , M11 L437E, which contains mutations R47L, E64G, F81I, F87V, E143G, L188Q, E267V, G415S and L437E compared to wild‐type CYP102A1. This mutant appeared to selectively convert flucloxacillin to 5′‐HM‐FLX (Luirink et al ., ); 1 mL of a solution of 100 mM flucloxacillin in DMSO was added to 49 mL 100 mM potassium phosphate buffer pH 7.4 and incubated in the presence of 1 μM of M11 L437E and a NADPH‐regenerating system (NRS) for 20 h at room temperature with constant stirring. The NRS was consisted of 100 μM NADPH, 10 mM glucose‐6‐phosphate and 0.5 units·mL −1 glucose‐6‐phosphate dehydrogenase (final concentrations).…”