Site-directed mutagenesis of residues 164, 170, 171, 179, 220, 237 and 242 in PER-1 β-lactamase hydrolysing expanded-spectrum cephalosporins

Bouthors, Anne-Typhaine; Delettré, J.; Mugnier, Pierre; Jarlier, Vincent; Sougakoff, Wladimir

doi:10.1093/protein/12.4.313

Cited by 25 publications

(32 citation statements)

References 32 publications

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“…It involves the main chain oxygen atom of residue 104 in all structures except PER-1, and its contribution could evidently not be demonstrated by protein engineering in these enzymes. Replacement of Thr237 by an alanine residue increased the k cat /Km of the mutant protein on cephalosporin substrates by 10 to 100 fold (35). Interestingly, it is the reverse mutation, Ala237Thr, which improved hydrolysis on cephalosporin substrates in the TEM enzyme (67).…”

Section: Discussionmentioning

confidence: 99%

“…The PER-1 β-lactamase was expressed in E. coli and purified as previously described (34,35). Crystals were obtained using the vapour diffusion method : an 8 µl drop, made of 2 µl of protein (20 mg/ml in Tris/HCl 20 mM, DTT 0.5 mM, NaN3 0.06 %, pH 7.60) and of 6 µl of reservoir solution was equilibrated against 330 µl of reservoir solution containing 18% (v/v) saturated ammonium sulfate, 0.1 M sodium acetate (pH 4.7) and 1 mM dithiothreitol.…”

Section: Experimental Procedures Crystallizationmentioning

confidence: 99%

“…Surprisingly, site-directed mutagenesis on the PER-1 enzyme showed that none of the residues responsible for the cephalosporinase activity in the TEM and SHV families (104, 164, 179, 238, and 240) were implicated in the substrate profile of this enzyme (34,35). The 1.9 Å Xray structure of PER-1 presented in this report reveals a completely new fold of the Ω-loop region, so far one of the most conserved feature in the class A enzymes.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

Section: Experimental Procedures Crystallizationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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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“…Finally, protein engineering studies of PER-1 identified two residues whose mutations resulted in significant kinetic effects. First, the replacement of Thr104 with a Glu completely abolished the catalytic activity of the enzyme toward penicillins and reduced the k cat values for cephalosporins by a factor of 50 to 700 (193). According to the X-ray structure of PER-1, this mutation should disrupt the interaction of the ␥-hydroxyl group of Thr104 with Asn132.…”

Section: Structure-function Relationships Of Class a Enzymesmentioning

confidence: 99%

A Structure-Based Classification of Class A β-Lactamases, a Broadly Diverse Family of Enzymes

Slama²,

et al. 2016

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SUMMARYFor medical biologists, sequencing has become a commonplace technique to support diagnosis. Rapid changes in this field have led to the generation of large amounts of data, which are not always correctly listed in databases. This is particularly true for data concerning class A β-lactamases, a group of key antibiotic resistance enzymes produced by bacteria. Many genomes have been reported to contain putative β-lactamase genes, which can be compared with representative types. We analyzed several hundred amino acid sequences of class A β-lactamase enzymes for phylogenic relationships, the presence of specific residues, and cluster patterns. A clear distinction was first made between dd-peptidases and class A enzymes based on a small number of residues (S70, K73, P107, 130SDN132, G144, E166, 234K/R, 235T/S, and 236G [Ambler numbering]). Other residues clearly separated two main branches, which we named subclasses A1 and A2. Various clusters were identified on the major branch (subclass A1) on the basis of signature residues associated with catalytic properties (e.g., limited-spectrum β-lactamases, extended-spectrum β-lactamases, and carbapenemases). For subclass A2 enzymes (e.g., CfxA, CIA-1, CME-1, PER-1, and VEB-1), 43 conserved residues were characterized, and several significant insertions were detected. This diversity in the amino acid sequences of β-lactamases must be taken into account to ensure that new enzymes are accurately identified. However, with the exception of PER types, this diversity is poorly represented in existing X-ray crystallographic data.

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“…Similar trends have been observed in other TEM ESBL variants but are not limited to this family of enzymes. 3,23,[37][38][39] Thus, while selection was directed exclusively toward CTX hydrolysis, the evolution of this promiscuous binding function (CTX binding) 40 remained compatible with recognition of first-generation cephalosporins. Being relatively innocuous toward enzyme function, the additional mutations at positions 104, 105 and 240 indicate a degree of robustness in TEM-1, 41 which tolerated multiple active-site mutations and still allowed recognition of related compounds.…”

Section: Promiscuity Robustness and Plasticity In Substrate Bindingmentioning

confidence: 99%

High tolerance to simultaneous active‐site mutations in TEM‐1 β‐lactamase: Distinct mutational paths provide more generalized β‐lactam recognition

2008

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The diversity in substrate recognition spectra exhibited by various b-lactamases can result from one or a few mutations in the active-site area. Using Escherichia coli TEM-1 b-lactamase as a template that efficiently hydrolyses penicillins, we performed site-saturation mutagenesis simultaneously on two opposite faces of the active-site cavity. Residues 104 and 105 as well as 238, 240, and 244 were targeted to verify their combinatorial effects on substrate specificity and enzyme activity and to probe for cooperativity between these residues. Selection for hydrolysis of an extended-spectrum cephalosporin, cefotaxime (CTX), led to the identification of a variety of novel mutational combinations. In vivo survival assays and in vitro characterization demonstrated a general tendency toward increased CTX and decreased penicillin resistance. Although selection was undertaken with CTX, productive binding (K M ) was improved for all substrates tested, including benzylpenicillin for which catalytic turnover (k cat ) was reduced. This indicates broadened substrate specificity, resulting in more generalized (or less specialized) variants. In most variants, the G238S mutation largely accounted for the observed properties, with additional mutations acting in an additive fashion to enhance these properties. However, the most efficient variant did not harbor the mutation G238S but combined two neighboring mutations that acted synergistically, also providing a catalytic generalization. Our exploration of concurrent mutations illustrates the high tolerance of the TEM-1 active site to multiple simultaneous mutations and reveals two distinct mutational paths to substrate spectrum diversification.

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Site-directed mutagenesis of residues 164, 170, 171, 179, 220, 237 and 242 in PER-1 β-lactamase hydrolysing expanded-spectrum cephalosporins

Cited by 25 publications

References 32 publications

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

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

A Structure-Based Classification of Class A β-Lactamases, a Broadly Diverse Family of Enzymes

High tolerance to simultaneous active‐site mutations in TEM‐1 β‐lactamase: Distinct mutational paths provide more generalized β‐lactam recognition

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