PDBsum entry 1h2g

Hydrolase

PDB id: 1h2g

Contents

Protein chains

205 a.a. *

557 a.a. *

Ligands

EDO ×4

Metals

_CA

Waters ×596

* Residue conservation analysis

PDB id:

1h2g

Name:

Hydrolase

Title:

Altered substrate specificity mutant of penicillin acylase

Structure:

Penicillin g acylase alpha subunit. Chain: a. Synonym: penicillin amidohydrolase, penicillin g amidase. Engineered: yes. Penicillin g acylase beta subunit. Chain: b. Synonym: penicillin amidohydrolase, penicillin g amidase. Engineered: yes. Mutation: yes.

Source:

Escherichia coli. Organism_taxid: 562. Atcc: 11105. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Biol. unit:

Dimer (from PDB file)

Resolution:

2.00Å R-factor: 0.152 R-free: 0.194

Authors:

C.E.Mcvey,M.Morillas,J.A.Brannigan,A.G.Ladurner,L.J.Forney,R.Virden

Key ref:

M.Morillas et al. (2003). Mutations of penicillin acylase residue B71 extend substrate specificity by decreasing steric constraints for substrate binding. Biochem J, 371, 143-150. PubMed id: 12511194

Date:

08-Aug-02 Release date: 17-Jul-03

Protein chain

P06875  (PAC_ECOLX) -  Penicillin G acylase from Escherichia coli

Seq: 846 a.a.

Struc: 205 a.a.

Protein chain

P06875  (PAC_ECOLX) -  Penicillin G acylase from Escherichia coli

Seq: 846 a.a.

Struc: 557 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: Chains A, B: E.C.3.5.1.11 - penicillin amidase.
• Pathway: Penicillin Biosynthesis and Metabolism
• Reaction: a penicillin + H2O = 6-aminopenicillanate + a carboxylate

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

Biochem J 371:143-150 (2003)

PubMed id: 12511194

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

M.Morillas, C.E.McVey, J.A.Brannigan, A.G.Ladurner, L.J.Forney, R.Virden.

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.