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 (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 K M = 11.0 and 8.1 mM, respectively.

Protein Engineering of d‐Succinylase from Cupriavidus sp. for d‐Amino Acid Synthesis and the Structural Implications

“…However, it was difficult to determine the target amino acid of the mutation from such limited information. Although many crystal structures of these acylases had been determined, [13–16] there was almost no structural information regarding enantioselectivity. Therefore, we constructed a screening system using semirandom mutagenesis by error‐prone PCR as shown in Scheme 2.…”

Hypothesis: The electrical properties of coronavirus

“…Decades of research into protein-protein interactions (PPIs) have been studied by many researchers such as biophysics have produced a remarkable summary knowledge of the protein interactions (Bagher et al, 2019;Ferrario et al 2019;Gilson and Zhou 2007;Kundrotas and Alexov 2006;Murray et al 1998;Zhou and Gilson 2009). Some works have focused on the role of the electrostatic forces in protein-protein recognition.…”

Strategy for the Biosynthesis of Short Oligopeptides: Green and Sustainable Chemistry

“…Considering the nature of the similar chemical structures of glutaryl-7-ACA, adipyl-7-ADCA, and cephalosporin C, attempts have been made to create mutants of cephalosporin acylases with improved activities toward adipyl-7-ADCA and cephalosporin C. From this perspective, engineering modifications of cephalosporin acylase would be a feasible strategy to achieve this goal, and significant progress has been made in addressing the concerns of low substrate specificity, substrate inhibition, and product inhibition encountered in practice [104,105].…”