PDBsum entry 3ixh

Hydrolase

PDB id: 3ixh

Contents

Protein chains

358 a.a. *

Ligands

CEF

Waters ×182

* Residue conservation analysis

PDB id:

3ixh

Name:

Hydrolase

Title:

X-ray crystal structure of the extended-spectrum ampc y221g mutant beta-lactamase in complex with cefotaxime at 2.3 angstrom resolution

Structure:

Beta-lactamase. Chain: b, a. Synonym: cephalosporinase. Engineered: yes. Mutation: yes

Source:

Escherichia coli. Organism_taxid: 83333. Strain: k12. Gene: ampa, ampc, b4150, jw4111. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.30Å R-factor: 0.197 R-free: 0.248

Authors:

B.K.Shoichet,V.L.Thomas

Key ref:

V.L.Thomas et al. (2010). Structural bases for stability-function tradeoffs in antibiotic resistance. J Mol Biol, 396, 47-59. PubMed id: 19913034

Date:

04-Sep-09 Release date: 16-Feb-10

Protein chains

P00811  (AMPC_ECOLI) -  Beta-lactamase from Escherichia coli (strain K12)

Seq: 377 a.a.

Struc: 358 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+)

J Mol Biol 396:47-59 (2010)

PubMed id: 19913034

Structural bases for stability-function tradeoffs in antibiotic resistance.

V.L.Thomas, A.C.McReynolds, B.K.Shoichet.

ABSTRACT

Preorganization of enzyme active sites for substrate recognition typically comes at a cost to the stability of the folded form of the protein; consequently, enzymes can be dramatically stabilized by substitutions that attenuate the size and preorganization "strain" of the active site. How this stability-activity tradeoff constrains enzyme evolution has remained less certain, and it is unclear whether one should expect major stability insults as enzymes mutate towards new activities or how these new activities manifest structurally. These questions are both germane and easy to study in beta-lactamases, which are evolving on the timescale of years to confer resistance to an ever-broader spectrum of beta-lactam antibiotics. To explore whether stability is a substantial constraint on this antibiotic resistance evolution, we investigated extended-spectrum mutants of class C beta-lactamases, which had evolved new activity versus third-generation cephalosporins. Five mutant enzymes had between 100-fold and 200-fold increased activity against the antibiotic cefotaxime in enzyme assays, and the mutant enzymes all lost thermodynamic stability (from 1.7 kcal mol(-)(1) to 4.1 kcal mol(-)(1)), consistent with the stability-function hypothesis. Intriguingly, several of the substitutions were 10-20 A from the catalytic serine; the question of how they conferred extended-spectrum activity arose. Eight structures, including complexes with inhibitors and extended-spectrum antibiotics, were determined by X-ray crystallography. Distinct mechanisms of action, including changes in the flexibility and ground-state structures of the enzyme, are revealed for each mutant. These results explain the structural bases for the antibiotic resistance conferred by these substitutions and their corresponding decrease in protein stability, which will constrain the evolution of new antibiotic resistance.