PDBsum entry: 2bg7

Hydrolase

PDB id: 2bg7

Contents

Protein chains

217 a.a. *

Ligands

GOL ×12

SO4 ×4

Metals

_ZN ×2

Waters ×348

* Residue conservation analysis

PDB id:

2bg7

Name:

Hydrolase

Title:

Bacillus cereus metallo-beta-lactamase (bcii) arg (121) cys mutant. Solved at ph4.5 using 20 micromolar znso4 in the buffer. 1mm dtt was used as a reducing agent. Cys221 is oxidized.

Structure:

Beta-lactamase ii. Chain: a, b. Synonym: metallo-beta-lactamase, penicillinase, cephalosporinase. Engineered: yes. Mutation: yes

Source:

Bacillus cereus. Organism_taxid: 1396. Strain: 569/h/9. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

2.10Å R-factor: 0.190 R-free: 0.222

Authors:

A.M.Davies,R.M.Rasia,A.J.Vila,B.J.Sutton,S.M.Fabiane

Key ref:

A.M.Davies et al. (2005). Effect of pH on the active site of an Arg121Cys mutant of the metallo-beta-lactamase from Bacillus cereus: implications for the enzyme mechanism. Biochemistry, 44, 4841-4849. PubMed id: 15779910 DOI: 10.1021/bi047709t

Date:

17-Dec-04 Release date: 31-Mar-05

Protein chains

P04190  (BLA2_BACCE) -  Beta-lactamase 2

Seq: 257 a.a.

Struc: 217 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

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: 10.1021/bi047709t

Biochemistry 44:4841-4849 (2005)

PubMed id: 15779910

Effect of pH on the active site of an Arg121Cys mutant of the metallo-beta-lactamase from Bacillus cereus: implications for the enzyme mechanism.

A.M.Davies, R.M.Rasia, A.J.Vila, B.J.Sutton, S.M.Fabiane.

ABSTRACT

The zinc-dependent metallo-beta-lactamases are a group of bacterial enzymes that pose a threat to the future efficacy of present-day antibiotics. Their mechanism is poorly understood, and there are no clinically useful inhibitors. While most members of the group contain two tightly bound zinc ions in their active sites, the Bacillus cereus enzyme has a much lower affinity for its second zinc (Zn2), thought to be due to the presence of Arg121 immediately beneath the floor of the active site (cf. Cys/Ser/His121 in the bizinc enzymes). Crystal structures of the Arg121Cys mutant of the B. cereus 569/H/9 enzyme were solved at pH 7.0, 5.0, and 4.5, each in the presence of either 20 microM or 20 mM Zn(2+) to generate the mono- and bizinc forms, respectively. Surprisingly, the structure of the active site was unaffected by the mutation; a network of ordered water molecules replaced the interactions made by the arginine side chain, and the occupancy of Zn2 appeared minimally changed. As the pH was lowered, Zn2 moved away from one of its ligands, Asp120, but was "tracked" by two others, Cys221 and His263. Furthermore, the hydroxide ion (and proposed nucleophile for beta-lactam hydrolysis) was bound to Zn1 at pH 5 and above but absent at pH 4.5. This provides experimental evidence for an earlier proposed mechanism in which protonation of Asp120 and the Zn1-bound hydroxide are the two events that lead to the loss of activity at low pH.