Modelling of substrate access and substrate binding to cephalosporin acylases.

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 (E86?, S369?, S460?) at the entrance to the binding pocket, preventing substrate access. A second non-productive binding site (G372?, W376?, L457?) 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.

SUBMITTER: Ferrario V 

PROVIDER: S-EPMC6712217 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature