1H2G

Altered substrate specificity mutant of penicillin acylase

Summary for 1H2G

• Entry DOI: 10.2210/pdb1h2g/pdb
• Related: 1AI4 1AI5 1AI6 1AI7 1AJN 1AJP 1AJQ 1CP9 1E3A 1FXH 1FXV 1GK9 1GKF 1GM7 1GM8 1GM9 1PNK 1PNL 1PNM
• Descriptor: PENICILLIN G ACYLASE ALPHA SUBUNIT, PENICILLIN G ACYLASE BETA SUBUNIT, 1,2-ETHANEDIOL, ... (5 entities in total)
• Functional Keywords: amidohydrolase, antibiotic resistance, altered specificity, zymogen, hydrolase
• Biological source: ESCHERICHIA COLI; More
• Cellular location: Periplasm: P06875 P06875
• Total number of polymer chains: 2
• Total formula weight: 86522.65

Authors

McVey, C.E.,Morillas, M.,Brannigan, J.A.,Ladurner, A.G.,Forney, L.J.,Virden, R. (deposition date: 2002-08-08, release date: 2003-07-17, Last modification date: 2023-12-13)

Primary citation

Morillas, M.,Mcvey, C.E.,Brannigan, J.A.,Ladurner, A.G.,Forney, L.J.,Virden, R.
Mutations of Penicillin Acylase Residue B71 Extend Substrate Specificity by Decreasing Steric Constraints for Substrate Binding
Biochem.J., 371:143-, 2003

PubMed 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.

PubMed: 12511194
DOI: 10.1042/BJ20021383

Experimental method

X-RAY DIFFRACTION (2 Å)