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

Tranier, S.; Bouthors, Anne-Typhaine; Maveyraud, Laurent; Guillet, Valérie; Sougakoff, Wladimir; Samama, Jean‐Pierre

doi:10.1074/jbc.m003802200

Cited by 61 publications

(66 citation statements)

References 67 publications

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“…The composition of the ⍀-loop (residues 161 to 179) differed considerably between subclass A1 and subclass A2 enzymes. Finally, as for PER-1 and PER-2, an alignment of subclass A2 ␤-lactamase sequences revealed the presence of several insertions (98,99).…”

Section: Primary Structure/sequence Analysismentioning

confidence: 83%

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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Section: Primary Structure/sequence Analysismentioning

confidence: 83%

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

Slama²,

et al. 2016

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“…Overall, one appreciates that the first common theme to emerge is that the active site is selectively "remodeled and expanded" to accommodate the bulky R 1 side chain of extendedspectrum cephalosporins [6]. This remodeling is observed in the atomic structures of Toho-1, TEM-52, TEM-64, the Gly238Ala ESBL in TEM, SHV-2, PER-1 and OXA-10 betalactamases [46][47][48][49][50][51][52]. Recently, the crystal structure of KPC-2 was determined, demonstrating modifications in the active site that allow access to carbapenems [53].…”

Section: Structural Properties Of Esblsmentioning

confidence: 99%

The continuing challenge of ESBLs

Pérez

Endimiani

Hujer

et al. 2007

Current Opinion in Pharmacology

265

209

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Summary of recent advancesSince their first description more than twenty years ago, Escherichia coli and Klebsiella pneumoniae possessing extended-spectrum class A beta-lactamases (ESBLs) continue to thwart our best clinical efforts. In the "early years" the most common beta-lactamases were of the TEM and SHV varieties. Now, CTX-M enzymes are being discovered though out the world and are becoming the most prevalent beta-lactamases found in clinical isolates. The Klebsiella pneumoniae carbapenemases (KPC) (ESBL type enzymes that confer resistance to extended spectrum cephalosporins and carbapenems) present the most significant challenge to date. Structural studies of ESBLs indicate that active site expansion and remodeling are responsible for this extended hydrolytic activity. Continuing questions still exist regarding the optimal detection method for ESBLs. Most relevant are the increasing concerns regarding the status of carbapenems as "best therapy" for ESBL producing bacteria in light of the emergence of carbapenemases.

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“…Finally, a hydrogen bond is formed between the Ser61 hydroxyl group O γ and the Ser122 hydroxyl group O γ (3.02 Ǻ). For class A β-lactamases, distances of 3.17 Å for PER-1 (1e25), 15 3.15 A for TEM-1 in 1.55 Å resolution structure (1zg4), 16 but 3.55 Å in 1.8 Å resolution structure (1btl), 17 and 2.89 Å in Toho-1 (1iys) 18 are found. In class A β-lactamases, the Lys73 δ-NH 2 group is hydrogen bonded to the carboxylate group of Glu166.…”

Section: Active Sitementioning

confidence: 97%

“…6a). In particular, the cis peptide bond between residues 166 and 167, an invariant feature of class A β-lactamases, except in PER-1, which displays a different Ω-loop fold, 15 is observed in position 159 of PBP-A. On the other hand, three invariable electrostatic interactions maintain the loop integrity in class A β-lactamases: a hydrogen bond between the side chains of Lys73 and Glu166, a salt bridge between Arg164 and Asp179, and hydrogen bonds between the CO and NH2 groups of Asn136 side chain and the mainchain NH and CO groups of Glu166, respectively.…”

Section: Comparison With Tem-1 and Per-1 β-Lactamasesmentioning

confidence: 99%

Structure of PBP-A from Thermosynechococcus elongatus, a Penicillin-Binding Protein Closely Related to Class A β-Lactamases

Urbach¹,

Evrard²,

Pudzaitis³

et al. 2009

Journal of Molecular Biology

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Molecular evolution has always been a subject of discussions, and researchers are interested in understanding how proteins with similar scaffolds can catalyze different reactions. In the superfamily of serine penicillinrecognizing enzymes, D-alanyl-D-alanine peptidases and β-lacta-mases are phylogenetically linked but feature large differences of reactivity towards their respective substrates. In particular, while β-lactamases hydrolyze penicillins very fast, leading to their inactivation, these molecules inhibit D-alanyl-D-alanine peptidases by forming stable covalent penicilloyl enzymes. In cyanobacteria, we have discovered a new family of penicillinbinding proteins (PBPs) presenting all the sequence features of class A β-lactamases but having a six-amino-acid deletion in the conserved Ω-loop and lacking the essential Glu166 known to be involved in the penicillin hydrolysis mechanism. With the aim of evolving a member of this family into a β-lactamase, PBP-A from Thermosynechococcus elongatus has been chosen because of its thermostability. Based on sequence alignments, introduction of a glutamate in position 158 of the shorter Ω-loop afforded an enzyme with a 50-fold increase in the rate of penicillin hydrolysis. The crystal structures of PBP-A in the free and penicilloylated forms at 1.9 Å resolution and of L158E mutant at 1.5 Å resolution were also solved, giving insights in the catalytic mechanism of the proteins. Since all the active-site elements of PBP-A-L158E, including an essential water molecule, are almost perfectly superimposed with those of a class A β-lactamase such as TEM-1, the question why our mutant is still 5 orders of magnitude less active as a penicillinase remains and our results emphasize how far we are from understanding the secrets of enzymes. Based on the few minor differences between the active sites of PBP-A and TEM-1, mutations were introduced in the L158E enzyme, but while activities on D-Ala-D-Ala mimicking substrates were severely impaired, further improvement in penicillinase activity was unsuccessful.

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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

Cited by 61 publications

References 67 publications

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

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

The continuing challenge of ESBLs

Structure of PBP-A from Thermosynechococcus elongatus, a Penicillin-Binding Protein Closely Related to Class A β-Lactamases

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