Mutations of penicillin acylase residue B71 extend substrate specificity by decreasing steric constraints for substrate binding

Abstract

Two mutant forms of penicillin acylase from Escherichia coli strains, selected using directed evolution for the ability to use glutaryl-L-leucine for growth [Forney, Wong and Ferber (1989) Appl. Environ. Microbiol. 55, 2550-2555], are changed within one codon, replacing the B-chain residue Phe(B71) with either Cys or Leu. Increases of up to a factor of ten in k (cat)/ K (m) values for substrates possessing a phenylacetyl leaving group are consistent with a decrease in K (s). Values of k (cat)/ K (m) for glutaryl-L-leucine are increased at least 100-fold. A decrease in k (cat)/ K (m) for the Cys(B71) mutant with increased pH is consistent with binding of the uncharged glutaryl group. The mutant proteins are more resistant to urea denaturation monitored by protein fluorescence, to inactivation in the presence of substrate either in the presence of urea or at high pH, and to heat inactivation. The crystal structure of the Leu(B71) mutant protein, solved to 2 A resolution, shows a flip of the side chain of Phe(B256) into the periphery of the catalytic centre, associated with loss of the pi-stacking interactions between Phe(B256) and Phe(B71). Molecular modelling demonstrates that glutaryl-L-leucine may bind with the uncharged glutaryl group in the S(1) subsite of either the wild-type or the Leu(B71) mutant but with greater potential freedom of rotation of the substrate leucine moiety in the complex with the mutant protein. This implies a smaller decrease in the conformational entropy of the substrate on binding to the mutant proteins and consequently greater catalytic activity.

Improved β-lactam acylases and their use as industrial biocatalysts

“…In contrast to these results, growth selection of mutants of E. coli PGA on glutaryl-leucine resulted in variants with an improved interaction with the leucine moiety of the selection substrate, but no activity towards the corresponding b-lactam compound, glutaryl-7-ACA [22].…”

Circular dichroism analysis of penicillin G acylase covalently immobilized on silica nanoparticles

“…Table 1 gives estimates of the secondary structure element fractions for the dissolved (free) penicillin G acylase together with the fractions obtained from its crystal structure [34] (PDB code for penicillin G acylase: 1h2g). The DSSP method by Kabsch and Sander [35] was used to extract the secondary structures of the protein from the coordinates determined by X-ray crystallography.…”

Engineering the Substrate Specificity of a Thermophilic Penicillin Acylase from Thermus thermophilus

“…Due to the industrial interest in PGA, mutational studies have been performed for over 20 years with the aim of altering its thermostability (23,24) and substrate specificity (25)(26)(27)(28)(29)(30)(31). To date, crystal structures of E. coli (32), P. rettgeri (28), and A. faecalis (33) PGAs are available, which have contributed either to rational design or to the explanation of results obtained by random mutagenesis techniques.…”