Abstract
Semisynthetic cephalosporins are widely used antibiotics currently produced by different chemical steps under harsh conditions, which results in a considerable amount of toxic waste. Biocatalytic synthesis by the cephalosporin acylase from Pseudomonas sp. strain N176 is a promising alternative. Despite intensive engineering of the enzyme, the catalytic activity is still too low for a commercially viable process. To identify the bottlenecks which limit the success of protein engineering efforts, a series of MD simulations was performed to study for two acylase variants (WT, M6) the access of the substrate cephalosporin C from the bulk to the active site and the stability of the enzyme-substrate complex. In both variants, cephalosporin C was binding to a non-productive substrate binding site (E86α, S369β, S460β) at the entrance to the binding pocket, preventing substrate access. A second non-productive binding site (G372β, W376β, L457β) was identified within the binding pocket, which competes with the active site for substrate binding. Noteworthy, substrate binding to the protein surface followed a Langmuir model resulting in binding constants K = 7.4 and 9.2 mM for WT and M6, respectively, which were similar to the experimentally determined Michaelis constants KM = 11.0 and 8.1 mM, respectively.
Highlights
Semisynthetic cephalosporins are widely used antibiotics to protect against extended-spectrum β-lactamase producing pathogens
Two different series of molecular dynamics (MD) simulations were performed to compare the binding of the substrate cephalosporin C (CPC) to two variants of cephalosporin acylase (CA) from Pseudomonas sp
In a second series of simulations, the orientation and position of a CPC molecule in the substrate binding pocket of CA was modelled starting from an enzyme-substrate complex, where the CPC substrate was placed in a productive binding pose, corresponding to the Near Attack Conformation (NAC)[25,26,27] (Fig. S1A)
Summary
Semisynthetic cephalosporins are widely used antibiotics to protect against extended-spectrum β-lactamase producing pathogens. Because of its industrial potential for cephalosporin production, intensive research efforts have been devoted to improve the catalytic activity of the class III enzyme from Pseudomonas sp. Such a design strategy misses two important bottlenecks that might limit catalytic activity: the presence of non-productive substrate binding poses (meaning binding poses not compatible with the catalytic mechanism) which compete with productive binding[23], and the access of substrate from the bulk to the active site[24] To address those possible limitations, molecular dynamics (MD) simulations were performed and two enzyme variants were compared: M31βF (WT) and M31βF/F58βN/H70βS/I176βT (M6)[20]. The simulations were analyzed to identify non-productive binding sites in the enzyme binding pocket and bottlenecks upon substrate access
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