Extended-spectrum and inhibitor-resistant TEM-type beta-lactamases: mutations, specificity, and three-dimensional structure

Knox, James R.

doi:10.1128/aac.39.12.2593

Cited by 296 publications

(351 citation statements)

References 88 publications

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“…In conclusion, we present evidence that TEM and SHV are different in their intrinsic binding and turnover of inhibitors (12). Despite similarities in sequence and phenotype between TEM and SHV, the kinetic differences revealed herein suggest that these two class A ␤-lactamases follow unique pathways in response to antibiotic pressure (an argument that would have significant implications for the comparative study of penicillininactivating enzymes among prokaryotes).…”

Section: Discussionmentioning

confidence: 87%

Probing Active Site Chemistry in SHV β-Lactamase Variants at Ambler Position 244

Thomson

Distler

Prati

et al. 2006

Journal of Biological Chemistry

109

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Inhibitor-resistant class A ␤-lactamases are an emerging threat to the use of ␤-lactam/␤-lactamase inhibitor combinations (e.g. amoxicillin/clavulanate) in the treatment of serious bacterial infections. In the TEM family of Class A ␤-lactamases, single amino acid substitutions at Arg-244 confer resistance to clavulanate inactivation. To understand the amino acid sequence requirements in class A ␤-lactamases that confer resistance to clavulanate, we performed site-saturation mutagenesis of Arg-244 in SHV-1, a related class A ␤-lactamase found in Klebsiella pneumoniae. Twelve SHV enzymes with amino acid substitutions at Arg-244 resulted in significant increases in minimal inhibitory concentrations to ampicillin/ clavulanate when expressed in Escherichia coli. Kinetic analyses of SHV-1, R244S, R244Q, R244L, and R244E ␤-lactamases revealed that the main determinant of clavulanate resistance was reduced inhibitor affinity. In contrast to studies in the highly similar TEM enzyme, we observed increases in clavulanate k inact for all mutants. Electrospray ionization mass spectrometry of clavulanate inhibited SHV-1 and R244S showed nearly identical mass adducts, arguing against a difference in the inactivation mechanism. Testing a wide range of substrates with C 3-4 carboxylates in different stereochemical orientations, we observed impaired affinity for all substrates among inhibitor resistant variants. Lastly, we synthesized two boronic acid transition state analogs that mimic cephalothin and found substitutions at Arg-244 markedly affect both the affinity and kinetics of binding to the chiral, deacylation transition state inhibitor. These data define a role for Arg-244 in substrate and inhibitor binding in the SHV ␤-lactamase.␤-Lactams have been the cornerstone of antibacterial chemotherapy since the introduction of penicillin. Although this family of antibiotics now contains numerous classes (penicillins, cephalosporins, and carbapenems, see Fig. 1), their use is threatened by the expansion and spread of ␤-lactamase enzymes (1). These bacterial enzymes hydrolyze ␤-lactam antibiotics before reaching their target, the penicillin-binding proteins. In an effort to retain the utility of several generations of life-saving ␤-lactam antibiotics, ␤-lactamase inhibitors (clavulanate, sulbactam, and tazobactam) have been developed. These mechanism-based inhibitors are ␤-lactam compounds that experience multistep reactions within the active site of ␤-lactamase enzymes (2-4). Typically the inhibitors lack antimicrobial activity and are formulated with a ␤-lactam antibiotic to act as shields against ␤-lactamase enzymes. This allows the ␤-lactam to bypass the ␤-lactamase and inactivate the penicillin binding proteins.Unfortunately, inhibitor-resistant class A ␤-lactamases are emerging in the clinic and undermining the use of ␤-lactam/␤-lactamase inhibitor therapy (5-7). Interest has been renewed in the discovery of novel ␤-lactamase inactivators to circumvent these inhibitor-resistant enzymes (8 -11). In this context, detailed analy...

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Section: Discussionmentioning

confidence: 87%

Probing Active Site Chemistry in SHV β-Lactamase Variants at Ambler Position 244

Thomson

Distler

Prati

et al. 2006

Journal of Biological Chemistry

109

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“…The third insertion occurs in a region where the sequence and the local fold were related to the extension of substrate profile in the TEM and SHV β-lactamases (28,32,33). In PER-1, four amino acids are inserted after residue 240 (Lys240a, Ala240b, Gly240c, and Lys240d).…”

Section: Insertionsmentioning

confidence: 99%

“…These enzymes display a very similar fold and the detailed comparisons of the structures were helpful in relating some by guest on May 9, 2018 http://www.jbc.org/ Downloaded from 5 significant differences in substrate profile to local structural features (23,24). The structural impact of point mutations leading to some 100 extended spectrum enzymes in the TEM and SHV families is less documented than the possible function of the invariant residues of the catalytic machinery (25)(26)(27)(28). Several studies supported the hypothesis that the cephalosporinase activity of these extended substrate enzymes was related to an increased flexibility of the Ω-loop region and to alterations of the S3 strand, two of the regions lining the active site (24,(29)(30)(31)(32)(33).…”

Section: Introductionmentioning

confidence: 99%

The High Resolution Crystal Structure for Class A β-Lactamase PER-1 Reveals the Bases for Its Increase in Breadth of Activity

Tranier¹,

Bouthors²,

Maveyraud³

et al. 2000

Journal of Biological Chemistry

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SUMMARYThe treatment of infectious diseases by β-lactam antibiotics is continuously challenged by the emergence and dissemination of new β-lactamases. In most cases, the cephalosporinase activity of class A enzymes results from a few mutations in the TEM and SHV penicillinases. The PER-1 β-lactamase was characterized as a class A enzyme displaying a cephalosporinase activity. This activity was however insensitive to the mutations of residues known to be critical for providing extended substrate profiles to TEM and SHV. The X-ray structure of the protein, solved at 1.9 Å resolution, reveals that two of the most conserved features in class A β-lactamase are not present in this enzyme: the fold of the Ω-loop and the cis conformation of the peptide bond between residues 166 and 167. The new fold of the Ω-loop and the insertion of four residues at the edge of strand S3 generate a broad cavity that may easily accomodate the bulky substituents of cephalosporin substrates. The trans conformation of the 166-167 bond is related to the presence of an aspartic acid at position 136. Selection of class A enzymes based on the occurrence of both Asp136 and Asn179 identifies a subgroup of enzymes with high sequence homology.

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“…Residues 237 and 104 are alanine and glutamate, respectively, in non-ESBL TEM and SHV enzymes. The Ala237Thr substitution in TEM enzymes improved their cephalosporinase activities, [29][30][31] and the Ser237Ala substitution decreased hydrolytic efficiency against oxyimino-cephalosporins in CTX-M-4 and Proteus vulgaris K1 b-lactamase, both ESBLs. 32,33 Ser237 and Asn104 interact with the carboxylate group of cefotaxime and the carbonyl group of the acylamide side-chain, respectively, in the acyl intermediate structure of Toho-1.…”

Section: Substrate Recognition and Catalysismentioning

confidence: 99%

Atomic Resolution Structures of CTX-M β-Lactamases: Extended Spectrum Activities from Increased Mobility and Decreased Stability

Chen

Delmas

Sirot

et al. 2005

Journal of Molecular Biology

135

190

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Extended-spectrum and inhibitor-resistant TEM-type beta-lactamases: mutations, specificity, and three-dimensional structure

Cited by 296 publications

References 88 publications

Probing Active Site Chemistry in SHV β-Lactamase Variants at Ambler Position 244

Probing Active Site Chemistry in SHV β-Lactamase Variants at Ambler Position 244

The High Resolution Crystal Structure for Class A β-Lactamase PER-1 Reveals the Bases for Its Increase in Breadth of Activity

Atomic Resolution Structures of CTX-M β-Lactamases: Extended Spectrum Activities from Increased Mobility and Decreased Stability

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