PDBsum entry 4f6h

Hydrolase

PDB id: 4f6h

Contents

Protein chain

223 a.a.

Ligands

PO4 ×2

SO4 ×2

Metals

_ZN

Waters ×178

PDB id:

4f6h

Name:

Hydrolase

Title:

Mutagenesis of zinc ligand residue cys221 reveals plasticity in the imp-1 metallo-b-lactamase active site

Structure:

Beta-lactamase. Chain: a. Synonym: beta-lactamase imp-1, bla-imp protein, extended-spectrum b- lactamase, imp-1 metallo-beta-lactmase, metallo beta lactamase, metallo-beta-lactamase, metallo-beta-lactamase imp-1, metallo-beta- lactamase blaimp-1. Engineered: yes. Mutation: yes

Source:

Pseudomonas aeruginosa. Organism_taxid: 287. Gene: blaimp-1, bla imp, bla-imp, blaesp, imp. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

1.74Å R-factor: 0.197 R-free: 0.226

Authors:

L.B.Horton,S.Shanker,B.Sankaran,R.Mikulski,N.G.Brown,K.Phillips, E.Lykissa,B.V.V.Prasad,T.G.Palzkill

Key ref:

L.B.Horton et al. (2012). Mutagenesis of zinc ligand residue Cys221 reveals plasticity in the IMP-1 metallo-β-lactamase active site. Antimicrob Agents Chemother, 56, 5667-5677. PubMed id: 22908171

Date:

14-May-12 Release date: 27-Mar-13

Protein chain

Q79MP6  (Q79MP6_PSEAI) -  Beta-lactamase from Pseudomonas aeruginosa

Seq: 246 a.a.

Struc: 223 a.a.*

* PDB and UniProt seqs differ at 7 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: Zn(2+)

Antimicrob Agents Chemother 56:5667-5677 (2012)

PubMed id: 22908171

Mutagenesis of zinc ligand residue Cys221 reveals plasticity in the IMP-1 metallo-β-lactamase active site.

L.B.Horton, S.Shanker, R.Mikulski, N.G.Brown, K.J.Phillips, E.Lykissa, B.V.Venkataram Prasad, T.Palzkill.

ABSTRACT

Metallo-β-lactamases catalyze the hydrolysis of a broad range of β-lactam antibiotics and are a concern for the spread of drug resistance. To analyze the determinants of enzyme structure and function, the sequence requirements for the subclass B1 IMP-1 β-lactamase zinc binding residue Cys221 were tested by saturation mutagenesis and evaluated for protein expression, as well as hydrolysis of β-lactam substrates. The results indicated that most substitutions at position 221 destabilized the enzyme. Only the enzymes containing C221D and C221G substitutions were expressed well in Escherichia coli and exhibited catalytic activity toward β-lactam antibiotics. Despite the lack of a metal-chelating group at position 221, the C221G enzyme exhibited high levels of catalytic activity in the presence of exogenous zinc. Molecular modeling suggests the glycine substitution is unique among substitutions in that the complete removal of the cysteine side chain allows space for a water molecule to replace the thiol and coordinate zinc at the Zn2 zinc binding site to restore function. Multiple methods were used to estimate the C221G Zn2 binding constant to be 17 to 43 μM. Studies of enzyme function in vivo in E. coli grown on minimal medium showed that both IMP-1 and the C221G mutant exhibited compromised activity when zinc availability was low. Finally, substitutions at residue 121, which is the IMP-1 equivalent of the subclass B3 zinc-chelating position, failed to rescue C221G function, suggesting the coordination schemes of subclasses B1 and B3 are not interchangeable.