PDBsum entry 1k5q

Hydrolase

PDB id: 1k5q

Contents

Protein chains

206 a.a. *

557 a.a. *

Ligands

PAC

Metals

_CA

Waters ×358

* Residue conservation analysis

PDB id:

1k5q

Name:

Hydrolase

Title:

Penicillin acylase, mutant complexed with paa

Structure:

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

Source:

Escherichia coli. Organism_taxid: 562. Gene: pac. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PQS)

Resolution:

2.34Å R-factor: 0.166 R-free: 0.218

Authors:

C.M.H.Hensgens,E.Keizer,H.J.Snijder,B.W.Dijkstra

Key ref:

W.B.Alkema et al. (2004). Structural and kinetic studies on ligand binding in wild-type and active-site mutants of penicillin acylase. Protein Eng Des Sel, 17, 473-480. PubMed id: 15254299

Date:

12-Oct-01 Release date: 02-Sep-03

Protein chain

P06875  (PAC_ECOLX) -  Penicillin G acylase from Escherichia coli

Seq: 846 a.a.

Struc: 206 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 2 residue positions (black crosses)

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

penicillin + H2O = 6-aminopenicillanate (Bound ligand (Het Group name = PAC) matches with 41.18% similarity) + carboxylate

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

reference

Protein Eng Des Sel 17:473-480 (2004)

PubMed id: 15254299

Structural and kinetic studies on ligand binding in wild-type and active-site mutants of penicillin acylase.

W.B.Alkema, C.M.Hensgens, H.J.Snijder, E.Keizer, B.W.Dijkstra, D.B.Janssen.

ABSTRACT

Penicillin acylase catalyses the condensation of Calpha-substituted phenylacetic acids with beta-lactam nucleophiles, producing semi-synthetic beta-lactam antibiotics. For efficient synthesis a low affinity for phenylacetic acid and a high affinity for Calpha-substituted phenylacetic acid derivatives is desirable. We made three active site mutants, alphaF146Y, betaF24A and alphaF146Y/betaF24A, which all had a 2- to 10-fold higher affinity for Calpha-substituted compounds than wild-type enzyme. In addition, betaF24A had a 20-fold reduced affinity for phenylacetic acid. The molecular basis of the improved properties was investigated by X-ray crystallography. These studies showed that the higher affinity of alphaF146Y for (R)-alpha-methylphenylacetic acid can be explained by van der Waals interactions between alphaY146:OH and the Calpha-substituent. The betaF24A mutation causes an opening of the phenylacetic acid binding site. Only (R)-alpha-methylphenylacetic acid, but not phenylacetic acid, induces a conformation with the ligand tightly bound, explaining the weak binding of phenylacetic acid. A comparison of the betaF24A structure with other open conformations of penicillin acylase showed that betaF24 has a fixed position, whereas alphaF146 acts as a flexible lid on the binding site and reorients its position to achieve optimal substrate binding.