PDBsum entry 1djc

Hydrolase

PDB id: 1djc

Contents

Protein chain

257 a.a. *

Ligands

SO4 ×3

Waters ×426

* Residue conservation analysis

PDB id:

1djc

Name:

Hydrolase

Title:

Structure of beta-lactamase precursor, s70a mutant, at 120k

Structure:

Beta-lactamase. Chain: a. Synonym: penicillinase. Engineered: yes. Mutation: yes. Other_details: soaked with ampicillin but density was uninterpretable

Source:

Staphylococcus aureus. Organism_taxid: 1280. Strain: pc1. Gene: blaz. Expressed in: shuttle plasmid from escherichia coli mc1061 to staphylococcus aureus rn4220.

Biol. unit:

Dimer (from PQS)

Resolution:

2.00Å R-factor: 0.171

Authors:

C.C.H.Chen,O.Herzberg

Key ref:

C.C.Chen et al. (1996). Structure and kinetics of the beta-lactamase mutants S70A and K73H from Staphylococcus aureus PC1. Biochemistry, 35, 12251-12258. PubMed id: 8823158 DOI: 10.1021/bi961153v

Date:

13-Aug-96 Release date: 12-Mar-97

Protein chain

P00807  (BLAC_STAAU) -  Beta-lactamase from Staphylococcus aureus

Seq: 281 a.a.

Struc: 257 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+)

DOI no: 10.1021/bi961153v

Biochemistry 35:12251-12258 (1996)

PubMed id: 8823158

Structure and kinetics of the beta-lactamase mutants S70A and K73H from Staphylococcus aureus PC1.

C.C.Chen, T.J.Smith, G.Kapadia, S.Wäsch, L.E.Zawadzke, A.Coulson, O.Herzberg.

ABSTRACT

Two mutant beta-lactamases from Staphylococcus aureus PC1 which probe key catalytic residues have been produced by site-directed mutagenesis. In the S70A enzyme, the nucleophilic group that attacks the beta-lactam carbonyl carbon atom was eliminated. Consequently, the kcat values for hydrolysis of benzylpenicillin and nitrocefin have been reduced by 10(4)-10(5) compared with the wild-type enzyme. The crystal structure of S70A beta-lactamase has been determined at 2.1 A resolution. With the exception of the mutation site, the structure is identical to that of the native enzyme. The residual activity is attributed either to mistranslation that leads to production of wild-type enzyme and/or to remaining features of the active site that stabilize the tetrahedral transition state. Soaking of the crystals with ampicillin or clavulanate, followed by flash-freezing, has been carried out and the structures examined at 2.0 A resolution. For both experiments, the difference electron density maps revealed buildup of density in the active site that presumably corresponds to beta-lactam binding. However, neither electron density is sufficiently clear for defining the atomic details of the bound compounds. The K73H beta-lactamase has been prepared to test the possible role of Lys73 in proton transfer. It exhibits no detectable activity toward benzylpenicillin, and 10(5)-fold reduction of kcat for nitrocefin hydrolysis compared with the wild-type enzyme. No significant recovery of activity has been measured when the pH was varied between 5.0 and 8.0. The crystal structure of K73H beta-lactamase has been determined at 1.9 A resolution. While the overall structure is similar to that of the native enzyme, the electrostatic interactions between His73 and neighboring residues indicate that the imidazole ring is positively charged. In addition, the hydroxyl group of Ser70 adopts a position that is incompatible with nucleophilic attack on substrates. A crystal soaked with ampicillin was flash-frozen, and diffraction data were collected at 2.1 A resolution. The electron density map showed no indication of substrate binding.