PDBsum entry: 1htz

Hydrolase

PDB id: 1htz

Contents

Protein chains

(+ 0 more) 263 a.a. *

Waters ×585

* Residue conservation analysis

PDB id:

1htz

Name:

Hydrolase

Title:

Crystal structure of tem52 beta-lactamase

Structure:

Beta-lactamase mutant tem52. Chain: a, b, c, d, e, f. Engineered: yes

Source:

Klebsiella pneumoniae. Organism_taxid: 573. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693.

Resolution:

2.40Å R-factor: 0.217 R-free: 0.261

Authors:

R.C.Stevens,M.C.Orencia

Key ref:

M.C.Orencia et al. (2001). Predicting the emergence of antibiotic resistance by directed evolution and structural analysis. Nat Struct Biol, 8, 238-242. PubMed id: 11224569 DOI: 10.1038/84981

Date:

03-Jan-01 Release date: 21-Mar-01

Protein chains

Q9R435  (Q9R435_KLEPN) -  Extended spectrum beta-lactamase

Seq: 286 a.a.

Struc: 263 a.a.

 Gene Ontology (GO) functional annotation 

• Biological process: response to antibiotic (2 terms)
• Biochemical function: hydrolase activity (2 terms)

DOI no: 10.1038/84981

Nat Struct Biol 8:238-242 (2001)

PubMed id: 11224569

Predicting the emergence of antibiotic resistance by directed evolution and structural analysis.

M.C.Orencia, J.S.Yoon, J.E.Ness, W.P.Stemmer, R.C.Stevens.

ABSTRACT

Directed evolution can be a powerful tool to predict antibiotic resistance. Resistance involves the accumulation of mutations beneficial to the pathogen while maintaining residue interactions and core packing that are critical for preserving function. The constraint of maintaining stability, while increasing activity, drastically reduces the number of possible mutational combination pathways. To test this theory, TEM-1 beta-lactamase was evolved using a hypermutator E. coli-based directed evolution technique with cefotaxime selection. The selected mutants were compared to two previous directed evolution studies and a database of clinical isolates. In all cases, evolution resulted in the generation of the E104K/M182T/G238S combination of mutations ( approximately 500-fold increased resistance), which is equivalent to clinical isolate TEM-52. The structure of TEM-52 was determined to 2.4 A. G238S widens access to the active site by 2.8 A whereas E104K stabilizes the reorganized topology. The M182T mutation is located 17 A from the active site and appears to be a global suppressor mutation that acts to stabilize the new enzyme structure. Our results demonstrate that directed evolution coupled with structural analysis can be used to predict future mutations that lead to increased antibiotic resistance.

Selected figure(s)

Figure 2.
Figure 2. Trends in mutation combinations and order of appearance (other mutations are observed, see Fig 1). In 1994, DNA shuffling predicted the mutation combination E104K/M182T/G238S as well as predicting mutations at positions 42 and 92 before their appearance in the clinic (these mutations were observed in 1998 (triple mutant), 1996 (position 42 mutant) and 1999 (position 92 mutant))1. The evolution of clinically isolated resistance mutants closely mimics the pathway described by the three directed evolution studies.

Figure 3.
Figure 3. Alternative views of the TEM-52 crystal structure. a, Nature and directed evolution experiments identified the E104K/M182T/G238S combination of mutations (green/red/green) and DNA shuffling predicted the loop mutations at positions 42, 92 and 241 (red). Catalytic residue positions are black and the B3 -strand is red. b, Overlay stereoview of wild type TEM-1 (cyan) and TEM-52 (blue) illustrating movement of loops 238 -243, 267 -271 and 40 -43. The G238S mutation (green) causes the Glu 240 side chain conformation to change (compare blue and purple side chains), which widens the active site by 2.8 Å, potentially allowing binding of bulky cephalosporins (catalytic Ser 70 side chain is shown in yellow). c, Stereoview of the electron density for the TEM-52 active site.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Struct Biol (2001, 8, 238-242) copyright 2001.

Figures were selected by an automated process.