PDBsum entry 2qjs

Hydrolase

PDB id: 2qjs

Contents

Protein chains

248 a.a. *

Metals

_ZN ×8

Waters ×396

* Residue conservation analysis

PDB id:

2qjs

Name:

Hydrolase

Title:

Stenotrophomonas maltophilia l1 metallo-beta-lactamase asp-120 asn mutant

Structure:

Metallo-beta-lactamase l1. Chain: a, b, c, d. Synonym: beta-lactamase type ii, penicillinase. Engineered: yes. Mutation: yes

Source:

Stenotrophomonas maltophilia. Organism_taxid: 40324. Strain: iid 1275. Gene: l1. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.25Å R-factor: 0.253 R-free: 0.283

Authors:

J.Crisp,R.Conners,J.Spencer

Key ref:

J.Crisp et al. (2007). Structural basis for the role of Asp-120 in metallo-beta-lactamases. Biochemistry, 46, 10664-10674. PubMed id: 17715946

Date:

09-Jul-07 Release date: 21-Aug-07

Protein chains

P52700  (BLA1_STEMA) -  Metallo-beta-lactamase L1 type 3 from Stenotrophomonas maltophilia

Seq: 290 a.a.

Struc: 248 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.3.5.2.6 - beta-lactamase.
• Pathway: Penicillin Biosynthesis and Metabolism
• Reaction: a beta-lactam + H2O = a substituted beta-amino acid
• Cofactor: Zn(2+)

Biochemistry 46:10664-10674 (2007)

PubMed id: 17715946

Structural basis for the role of Asp-120 in metallo-beta-lactamases.

J.Crisp, R.Conners, J.D.Garrity, A.L.Carenbauer, M.W.Crowder, J.Spencer.

ABSTRACT

Metallo-beta-lactamases (mbetals) are zinc-dependent enzymes that hydrolyze a wide range of beta-lactam antibiotics. The mbetal active site features an invariant Asp-120 that ligates one of the two metal ions (Zn2) and a metal-bridging water/hydroxide (Wat1). Previous studies show that substitutions at Asp-120 dramatically affect mbetal activity, but no consensus exists as to its role in beta-lactam turnover. Here we present crystal structures of the Asn and Cys mutants of Asp-120 of the L1 mbetal from Stenotrophomonas maltophilia. Both mutants retain a dinuclear zinc center with Wat1 present. In the essentially inactive Cys enzyme Zn2 is displaced to a more buried position relative to that in the wild-type enzyme. In the catalytically impaired Asn enzyme the coordination of Zn2 is altered, neither it nor Wat1 is coordinated by Asn-120, and the N-terminal 19 amino acids, important to cooperative interactions between subunits in the wild-type enzyme, are disordered. Comparison with the structure of L1 complexed with the hydrolyzed oxacephem moxalactam suggests that in the Cys mutant Zn2 can no longer make stabilizing interactions with anionic nitrogen species formed in the hydrolytic reaction. The diminished activity of the Asn mutant arises from a combination of loss of intersubunit interactions and impaired proton transfer to, and reduced interaction of Zn2 with, the substrate amide nitrogen. We conclude that, while interactions of Asp-120 with active site water molecules are important to proton transfer and possibly nucleophilic attack by Wat1, its primary role is to optimally position Zn2 for catalytically important interactions with the charged amide nitrogen of substrate.