Sequence and structure-based comparative analysis to assess, identify and improve the thermostability of penicillin G acylases

Abstract

Penicillin acylases are enzymes employed by the pharmaceutical industry for the manufacture of semi-synthetic penicillins. There is a continuous demand for thermostable and alkalophilic enzymes in such applications. We have carried out a computational analysis of known penicillin G acylases (PGAs) in terms of their thermostable nature using various protein-stabilizing factors. While the presence of disulfide bridges was considered initially to screen putative thermostable PGAs from the database, various other factors such as high arginine to lysine ratio, less content of thermolabile amino acids, presence of proline in β-turns, more number of ion-pair and other non-bonded interactions were also considered for comparison. A modified consensus approach designed could further identify stabilizing residue positions by site-specific comparison between mesostable and thermostable PGAs. A most likely thermostable enzyme identified from the analysis was PGA from Paracoccus denitrificans (PdPGA). This was cloned, expressed and tested for its thermostable nature using biochemical and biophysical experiments. The consensus site-specific sequence-based approach predicted PdPGA to be more thermostable than Escherichia coli PGA, but not as thermostable as the PGA from Achromobacter xylosoxidans. Experimental data showed that PdPGA was comparatively less thermostable than Achromobacter xylosoxidans PGA, although thermostability factors favored a much higher stability. Despite being mesostable, PdPGA being active and stable at alkaline pH is an advantage. Finally, several residue positions could be identified in PdPGA, which upon mutation selectively could improve the thermostability of the enzyme.

Exploring the Structurally Conserved Regions and Functional Significance in Bacterial N-Terminal Nucleophile (Ntn) Amide-Hydrolases

“…Factors such as the preference for Arginine over Lysine, the reduction in thermolabile amino acids, the increase in proline content, and the presence of stable ionic pairs are determining factors in this enhancement. Thermostability, influenced by the three-dimensional structure and amino acid sequence, is affected by elements such as the deamidation of Asparagine and Glutamine at high temperatures [40]. In comparing models from thermophilic organisms (6NVW and 8BRQ), they exhibit a lower abundance of proline, similar to positions in E. coli (1FXH), except in the loop region of SCR8B, which connects two folded beta strands.…”

Structure-based engineering of heparinase I with improved specific activity for degrading heparin

“…Using homology modeling, multiple sequence alignment, and site-directed mutagenesis, many enzymes have been modified to improve the activity, specificity and thermostability [17–20]. However, there are still no reports on the structure-based molecular evolution of HepI.…”