PDBsum entry: 1wup

Hydrolase

PDB id: 1wup

Contents

Protein chains

217 a.a. *

Ligands

ACY ×4

Metals

_ZN ×8

* Residue conservation analysis

PDB id:

1wup

Name:

Hydrolase

Title:

Crystal structure of metallo-beta-lactamase imp-1 mutant (d81e)

Structure:

Beta-lactamase imp-1. Chain: a, b, c, d. Synonym: imp-1 metallo-beta-lactamase, beta-lactamase, type ii, penicillinase, blaimp. Engineered: yes. Mutation: yes

Source:

Serratia marcescens. Organism_taxid: 615. Strain: tn9106. Gene: blaimp-1. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

3.00Å R-factor: 0.224 R-free: 0.295

Authors:

Y.Yamaguchi,Y.Yamagata,M.Goto

Key ref:

Y.Yamaguchi et al. (2005). Probing the role of Asp-120(81) of metallo-beta-lactamase (IMP-1) by site-directed mutagenesis, kinetic studies, and X-ray crystallography. J Biol Chem, 280, 20824-20832. PubMed id: 15788415 DOI: 10.1074/jbc.M414314200

Date:

08-Dec-04 Release date: 29-Mar-05

Protein chains

P52699  (BLAB_SERMA) -  Beta-lactamase IMP-1

Seq: 246 a.a.

Struc: 217 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: Zinc

 Gene Ontology (GO) functional annotation 

• Biological process: response to antibiotic (2 terms)
• Biochemical function: hydrolase activity (4 terms)

DOI no: 10.1074/jbc.M414314200

J Biol Chem 280:20824-20832 (2005)

PubMed id: 15788415

Probing the role of Asp-120(81) of metallo-beta-lactamase (IMP-1) by site-directed mutagenesis, kinetic studies, and X-ray crystallography.

Y.Yamaguchi, T.Kuroki, H.Yasuzawa, T.Higashi, W.Jin, A.Kawanami, Y.Yamagata, Y.Arakawa, M.Goto, H.Kurosaki.

ABSTRACT

Metallo-beta-lactamase IMP-1 is a di-Zn(II) metalloenzyme that efficiently hydrolyzes beta-lactam antibiotics. Wild-type (WT) IMP-1 has a conserved Asp-120(81) in the active site, which plays an important role in catalysis. To probe the catalytic role of Asp-120(81) in IMP-1, the IMP-1 mutants, D120(81)A and D120(81)E, were prepared by site-directed mutagenesis, and various kinetics studies were conducted. The IMP-1 mutants exhibited 10(2)-10(4)-fold drops in k(cat) values compared with WT despite the fact that they contained two Zn(II) ions in the active site. To evaluate the acid-base characteristics of Asp-120(81), the pH dependence for hydrolysis was examined by stopped-flow studies. No observable pK(a) values between pH 5 and 9 were found for WT and D120(81)A. The rapid mixing of equimolar amounts of nitrocefin and all enzymes failed to result in the detection of an anion intermediate of nitrocefin at 650 nm. These results suggest that Asp-120(81) of IMP-1 is not a factor in decreasing the pK(a) for the water bridging two Zn(II) ions and is not a proton donor to the anionic intermediate. In the case of D120(81)E, the nitrocefin hydrolysis product, which shows a maximum absorption at 460 nm, was bound to D120(81)E in the protonated form. The three-dimensional structures of D120(81)A and D120(81)E were also determined at 2.0 and 3.0 A resolutions, respectively. In the case of D120(81)E, the Zn-Zn distance was increased by 0.3 A compared with WT, due to the change in the coordination mode of Glu-120(81)OE1 and the positional shift in the conserved His-263(197) at the active site.

Selected figure(s)

Figure 1.
FIG. 1. Schematic representation of the active site of CcrA (PDB code 1ZNB [PDB] ) from B. fragilis (28). The residues are labeled according to the BBL standard numbering (20). The Zn(II) atoms are shown in green, oxygen atoms in red, nitrogen atom in blue, and sulfur atoms in yellow.

Figure 6.
FIG. 6. Plots of relative activity of nitrocefin hydrolysis by WT and the IMP-1 mutants D120(81)A and D120(81)E against the concentrations of phosphate ion at 30 °C.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2005, 280, 20824-20832) copyright 2005.

Figures were selected by an automated process.