Ultrahigh Resolution Structure of a Class A β-Lactamase: On the Mechanism and Specificity of the Extended-spectrum SHV-2 Enzyme

AbstractThe pathway for the biosynthesis of the bacterial cell wall is one of the most prolific antibiotic targets, exemplified by the widespread use of β-lactam antibiotics. Despite this, our structural understanding of class A penicillin binding proteins, which perform the last two steps in this pathway, is incomplete due to the inherent difficulty in their crystallization and the complexity of their substrates. Here, we determine the near atomic resolution structure of the 83 kDa class A PBP from Escherichia coli, PBP1b, using cryogenic electron microscopy and a styrene maleic acid anhydride membrane mimetic. PBP1b, in its apo form, is seen to exhibit a distinct conformation in comparison to Moenomycin-bound crystal structures. The work herein paves the way for the use of cryoEM in structure-guided antibiotic development for this notoriously difficult to crystalize class of proteins and their complex substrates.

Download Full-text

Effect of disulfide-bond introduction on the activity and stability of the extended-spectrum class A β-lactamase Toho-1

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics ◽

10.1016/j.bbapap.2006.06.004 ◽

2006 ◽

Vol 1764 (8) ◽

pp. 1349-1355 ◽

Cited By ~ 7

Author(s):

Akiko Shimizu-Ibuka ◽

Hiroshi Matsuzawa ◽

Hiroshi Sakai

Keyword(s):

Disulfide Bond ◽

Class A ◽

Extended Spectrum

Download Full-text

Characterization of beta-lactamase gene blaPER-2, which encodes an extended-spectrum class A beta-lactamase.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.3.616 ◽

1996 ◽

Vol 40 (3) ◽

pp. 616-620 ◽

Cited By ~ 82

Author(s):

A Bauernfeind ◽

I Stemplinger ◽

R Jungwirth ◽

P Mangold ◽

S Amann ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Beta Lactamase ◽

Reading Frame ◽

Beta Lactamases ◽

Class A ◽

Extended Spectrum ◽

The Family ◽

Extended Spectrum Beta Lactamases ◽

Lactamase Gene

Plasmidic extended-spectrum beta-lactamases of Ambler class A are mostly inactive against ceftibuten. Salmonella typhimurium JMC isolated in Argentina harbors a bla gene located on a plasmid (pMVP-5) which confers transferable resistance to oxyiminocephalosporins, aztreonam, and ceftibuten. The beta-lactamase PER-2 (formerly ceftibutenase-1; CTI-1) is highly susceptible to inhibition by clavulanate and is located at a pI of 5.4 after isoelectric focusing. The blaPER-2 gene was cloned and sequenced. The nucleotide sequence of a 2.2-kb insert in vector pBluescript includes an open reading frame of 927 bp. Comparison of the deduced amino acid sequence of PER-2 with those of other beta-lactamases indicates that PER-2 is not closely related to TEM or SHV enzymes (25 to 26% homology). PER-2 is most closely related to PER-1 (86.4% homology), an Ambler class A enzyme first detected in Pseudomonas aeruginosa. An enzyme with an amino acid sequence identical to that of PER-1, meanwhile, was found in various members of the family Enterobacteriaceae isolated from patients in Turkey. Our data indicate that PER-2 and PER-1 represent a new group of Ambler class A extended-spectrum beta-lactamases. PER-2 so far has been detected only in pathogens (S. typhimurium, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis) isolated from patients in South America, while the incidence of PER-1-producing strains so far has been restricted to Turkey, where it occurs both in members of the family Enterobacteriaceae and in P. aeruginosa.

Download Full-text

Frequency and diversity of Class A extended-spectrum β-lactamases in hospitals of the Auvergne, France: a 2 year prospective study

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkh395 ◽

2004 ◽

Vol 54 (3) ◽

pp. 634-639 ◽

Cited By ~ 41

Author(s):

C. De Champs ◽

C. Chanal ◽

D. Sirot ◽

R. Baraduc ◽

J. P. Romaszko ◽

...

Keyword(s):

Prospective Study ◽

Class A ◽

Extended Spectrum

Download Full-text

Mechanism of proton transfer in class A β-lactamase catalysis and inhibition by avibactam

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1922203117 ◽

2020 ◽

Vol 117 (11) ◽

pp. 5818-5825 ◽

Cited By ~ 5

Author(s):

Orville A. Pemberton ◽

Radwan E. Noor ◽

Vasantha Kumar M. V. ◽

Ruslan Sanishvili ◽

M. Trent Kemp ◽

...

Keyword(s):

Proton Transfer ◽

Critical Role ◽

Acid Dissociation ◽

Acid Dissociation Constant ◽

Sulfonate Group ◽

Enzyme Complex ◽

Ultrahigh Resolution ◽

Catalytic Residues ◽

Class A ◽

M 14

Gram-negative bacteria expressing class A β-lactamases pose a serious health threat due to their ability to inactivate all β-lactam antibiotics. The acyl–enzyme intermediate is a central milestone in the hydrolysis reaction catalyzed by these enzymes. However, the protonation states of the catalytic residues in this complex have never been fully analyzed experimentally due to inherent difficulties. To help unravel the ambiguity surrounding class A β-lactamase catalysis, we have used ultrahigh-resolution X-ray crystallography and the recently approved β-lactamase inhibitor avibactam to trap the acyl–enzyme complex of class A β-lactamase CTX-M-14 at varying pHs. A 0.83-Å-resolution CTX-M-14 complex structure at pH 7.9 revealed a neutral state for both Lys73 and Glu166. Furthermore, the avibactam hydroxylamine-O-sulfonate group conformation varied according to pH, and this conformational switch appeared to correspond to a change in the Lys73 protonation state at low pH. In conjunction with computational analyses, our structures suggest that Lys73 has a perturbed acid dissociation constant (pKa) compared with acyl–enzyme complexes with β-lactams, hindering its function to deprotonate Glu166 and the initiation of the deacylation reaction. Further NMR analysis demonstrated Lys73 pKato be ∼5.2 to 5.6. Together with previous ultrahigh-resolution crystal structures, these findings enable us to follow the proton transfer process of the entire acylation reaction and reveal the critical role of Lys73. They also shed light on the stability and reversibility of the avibactam carbamoyl acyl–enzyme complex, highlighting the effect of substrate functional groups in influencing the protonation states of catalytic residues and subsequently the progression of the reaction.

Download Full-text

Prevalence of Class A Extended-Spectrum β-Lactamases in Clinical Isolates of Acinetobacter baumannii and Pseudomonas aeruginosa

Annals of Laboratory Medicine ◽

10.3343/kjlm.2006.26.1.14 ◽

2006 ◽

Vol 26 (1) ◽

pp. 14-20 ◽

Cited By ~ 6

Author(s):

Se Jin Oh ◽

Sang Uk Lee ◽

Hyun Yong Hwang ◽

Il Kwon Bae ◽

Hyun Soo Jo ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Acinetobacter Baumannii ◽

Clinical Isolates ◽

Class A ◽

Extended Spectrum

Download Full-text

Acyl-intermediate Structures of the Extended-spectrum Class A β-Lactamase, Toho-1, in Complex with Cefotaxime, Cephalothin, and Benzylpenicillin

Journal of Biological Chemistry ◽

10.1074/jbc.m207884200 ◽

2002 ◽

Vol 277 (48) ◽

pp. 46601-46608 ◽

Cited By ~ 72

Author(s):

Tatsuro Shimamura ◽

Akiko Ibuka ◽

Shinya Fushinobu ◽

Takayoshi Wakagi ◽

Masaji Ishiguro ◽

...

Keyword(s):

Class A ◽

Extended Spectrum

Download Full-text

Genetic and biochemical characterization of GES-5, an extended-spectrum class A β-lactamase from Klebsiella pneumoniae

Diagnostic Microbiology and Infectious Disease ◽

10.1016/j.diagmicrobio.2007.02.013 ◽

2007 ◽

Vol 58 (4) ◽

pp. 465-468 ◽

Cited By ~ 37

Author(s):

Il Kwon Bae ◽

You-Nae Lee ◽

Seok Hoon Jeong ◽

Seong Geun Hong ◽

Jung Hun Lee ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Biochemical Characterization ◽

Class A ◽

Extended Spectrum

Download Full-text

Interactions of Ceftobiprole with β-Lactamases from Molecular Classes A to D

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00218-07 ◽

2007 ◽

Vol 51 (9) ◽

pp. 3089-3095 ◽

Cited By ~ 59

Author(s):

Anne Marie Queenan ◽

Wenchi Shang ◽

Malgosia Kania ◽

Malcolm G. P. Page ◽

Karen Bush

Keyword(s):

Functional Groups ◽

Hydrolytic Stability ◽

Enterobacter Cloacae ◽

Class A ◽

Extended Spectrum ◽

Class D ◽

The Family ◽

Class B ◽

The Common ◽

Antibacterial Spectrum

ABSTRACT The interactions of ceftobiprole with purified β-lactamases from molecular classes A, B, C, and D were determined and compared with those of benzylpenicillin, cephaloridine, cefepime, and ceftazidime. Enzymes were selected from functional groups 1, 2a, 2b, 2be, 2d, 2e, and 3 to represent β-lactamases from organisms within the antibacterial spectrum of ceftobiprole. Ceftobiprole was refractory to hydrolysis by the common staphylococcal PC1 β-lactamase, the class A TEM-1 β-lactamase, and the class C AmpC β-lactamase but was labile to hydrolysis by class B, class D, and class A extended-spectrum β-lactamases. Cefepime and ceftazidime followed similar patterns. In most cases, the hydrolytic stability of a substrate correlated with the MIC for the producing organism. Ceftobiprole and cefepime generally had lower MICs than ceftazidime for AmpC-producing organisms, particularly AmpC-overexpressing Enterobacter cloacae organisms. However, all three cephalosporins were hydrolyzed very slowly by AmpC cephalosporinases, suggesting that factors other than β-lactamase stability contribute to lower ceftobiprole and cefepime MICs against many members of the family Enterobacteriaceae.

Download Full-text