PDBsum entry 1jwp

Hydrolase

PDB id: 1jwp

Contents

Protein chain

263 a.a. *

Ligands

PO4

Waters ×291

* Residue conservation analysis

PDB id:

1jwp

Name:

Hydrolase

Title:

Structure of m182t mutant of tem-1 beta-lactamase

Structure:

Beta-lactamase tem. Chain: a. Fragment: tem-1. Engineered: yes. Mutation: yes

Source:

Escherichia coli. Organism_taxid: 562. Gene: bla. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.75Å R-factor: 0.186 R-free: 0.220

Authors:

X.Wang,G.Minasov,B.K.Shoichet

Key ref:

X.Wang et al. (2002). Evolution of an antibiotic resistance enzyme constrained by stability and activity trade-offs. J Mol Biol, 320, 85-95. PubMed id: 12079336 DOI: 10.1016/S0022-2836(02)00400-X

Date:

04-Sep-01 Release date: 05-Jun-02

Protein chain

P62593  (BLAT_ECOLX) -  Beta-lactamase TEM from Escherichia coli

Seq: 286 a.a.

Struc: 263 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: 10.1016/S0022-2836(02)00400-X

J Mol Biol 320:85-95 (2002)

PubMed id: 12079336

Evolution of an antibiotic resistance enzyme constrained by stability and activity trade-offs.

X.Wang, G.Minasov, B.K.Shoichet.

ABSTRACT

Pressured by antibiotic use, resistance enzymes have been evolving new activities. Does such evolution have a cost? To investigate this question at the molecular level, clinically isolated mutants of the beta-lactamase TEM-1 were studied. When purified, mutant enzymes had increased activity against cephalosporin antibiotics but lost both thermodynamic stability and kinetic activity against their ancestral targets, penicillins. The X-ray crystallographic structures of three mutant enzymes were determined. These structures suggest that activity gain and stability loss is related to an enlarged active site cavity in the mutant enzymes. In several clinically isolated mutant enzymes, a secondary substitution is observed far from the active site (Met182-->Thr). This substitution had little effect on enzyme activity but restored stability lost by substitutions near the active site. This regained stability conferred an advantage in vivo. This pattern of stability loss and restoration may be common in the evolution of new enzyme activity.

Selected figure(s)

Figure 3.
Figure 3. Comparing the in vivo stabilities of TEM-52 (E104K/M182T/G238S, left) and TEM-15 (E104K/G238S, right) at (a) 42 °C and (b) RT. Plates are representative of five independent assays for each mutant. The two top disks on each plate are CAZ and CTX from left to right, and the bottom disk is AM.

Figure 5.
Figure 5. A stereo view of the enlarged active sites of (a) G238A and (b) TEM-64 (E104K/R164S/M182T). (a) Superposition of the G238A (carbon atoms colored orange) and WT (magenta, PDB 1XPB[39.]) crystal structures (rms is 0.24 Å for all C^a atoms). A broken line indicates the putative steric clash (distance 2.96 Å) between the C^b atom of Ala238 and the carbonyl oxygen atom of Asn170 that would occur in WT. For G238A, carbon, nitrogen, and oxygen atoms are colored yellow, blue, and red, respectively. The catalytic water molecule in WT (magenta) and G238A (cyan) is shown. (b) Superposition of the C^a atom of TEM-64 (blue) and penicillin-bound TEM-1 E166N (orange, PDB 1FQG[33.]) crystal structures (rms is 0.85 Å for all C^a atoms). The penicillin G acyl-adduct is shown to identify the active site. The carbon atoms in penicillin G are colored green, nitrogen atoms blue, oxygen atoms red, and the sulfur atom yellow.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2002, 320, 85-95) copyright 2002.

Figures were selected by an automated process.