Critical involvement of a carbamylated lysine in catalytic function of class D β-lactamases

Golemi, Dasantila; Maveyraud, Laurent; Vakulenko, Sergei B.; Samama, Jean‐Pierre; Mobashery, Shahriar

doi:10.1073/pnas.241442898

Cited by 220 publications

(371 citation statements)

References 24 publications

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“…Carboxylation is favored at basic pH, but has been observed in crystal structures of the class D ␤-lactamases at as low as pH 6.0 (49) and perhaps even at pH 5.5 with low occupancy (40). The structures of apo-and acyl-BlaR S presented here were determined at pH 4.7 and 6.6, respectively.…”

Section: Role Of Lys 392 As the General Base In Acylation-mentioning

confidence: 81%

“…In either case, carboxylation of Lys 392 was not observed at any contour level in our electron density maps. In the class D ␤-lactamase structures, the non-carboxylated Lys 70 (corresponding to Lys 392 in BlaR1) seems to encourage an "inactive" conformation of Ser 115 (Ser 437 in BlaR1), in which the serine hydroxyl (presumed to shuttle a proton from the carboxylated lysine to the leaving group nitrogen of the ␤-lactam substrate) is somewhat displaced from its typical position in the active site (49,52). In contrast with many of the non-carboxylated structures of the class D ␤-lactamases, Kerff et al (19) noted that the active site of the apo form of the B. licheniformis BlaR1 sensor domain (which also lacks a carboxylated active-site lysine despite the fact that the crystals were grown at pH 7.0) closely resembles the "active" conformation.…”

Section: Role Of Lys 392 As the General Base In Acylation-mentioning

confidence: 99%

“…Mutation of either Pro 49 or Pro 52 to alanine in the L2 loop was observed to negate inducibility of ␤-lactamase expression (Table II). In fact, ␤-lactamase activity was not even detectable in the proline mutants because the cephaloridine concentrations were virtually identical to those of the ␤-lactamase-negative RN4220 control.…”

Section: Figmentioning

confidence: 99%

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Crystal Structures of the Apo and Penicillin-acylated Forms of the BlaR1 β-Lactam Sensor of Staphylococcus aureus

Wilke

Hills

Zhang

et al. 2004

Journal of Biological Chemistry

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Staphylococcus aureus is among the most prevalent and antibiotic-resistant of pathogenic bacteria. The resistance of S. aureus to prototypal ␤-lactam antibiotics is conferred by two mechanisms: (i) secretion of hydrolytic ␤-lactamase enzymes and (ii) production of ␤-lactam-insensitive penicillin-binding proteins (PBP2a). Despite their distinct modes of resistance, expression of these proteins is controlled by similar regulation systems, including a repressor (BlaI/MecI) and a multidomain transmembrane receptor (BlaR1/MecR1). Resistance is triggered in response to a covalent binding event between a ␤-lactam antibiotic and the extracellular sensor domain of BlaR1/MecR1 by transduction of the binding signal to an intracellular protease domain capable of repressor inactivation. This study describes the first crystal structures of the sensor domain of BlaR1 (BlaR S ) from S. aureus in both the apo and penicillin-acylated forms. The structures show that the sensor domain resembles the ␤-lactam-hydrolyzing class D ␤-lactamases, but is rendered a penicillin-binding protein due to the formation of a very stable acyl-enzyme. Surprisingly, conformational changes upon penicillin binding were not observed in our structures, supporting the hypothesis that transduction of the antibiotic-binding signal into the cytosol is mediated by additional intramolecular interactions of the sensor domain with an adjacent extracellular loop in BlaR1.The evolution and dissemination of antibiotic resistance in pathogenic bacteria are a growing concern. Many of the antimicrobial "wonder drugs" society has come to rely on for the treatment of bacterial infections have been neutralized by new strains equipped with a variety of molecular countermeasures. Methicillin-resistant Staphylococcus aureus is a prominent example of this (1-3). Although ␤-lactam antibiotics such as penicillin have long been the drugs of choice for infections of S. aureus, current therapies for methicillin-resistant S. aureus infections now depend on distinct antibiotic classes such as glycopeptides to counter the increased resistance to penicillin and its derivatives. The recent emergence of glycopeptide-resistant strains of S. aureus (4, 5) underscores the need not only for the development of novel therapeutics, but for better understanding of the molecular mechanisms involved in antibiotic resistance.␤-Lactam antibiotics kill bacteria by inhibiting the cell wall transpeptidases (also known as penicillin-binding proteins (PBPs) 1 ) that are responsible for the essential cross-linking of peptidoglycan during synthesis of the rigid bacterial cell wall. Resistance to ␤-lactam antibiotics in S. aureus can be conferred by two mechanisms. In one case, ␤-lactamase enzymes are secreted from the bacterium to hydrolytically inactivate ␤-lactam antibiotics. Resistance of this form is encoded by the blaZ gene and is carried by Ͼ95% of S. aureus isolates (3). The second resistance mechanism is expression of PBP2a, a cell wall transpeptidase with broad-spectrum insensitivity to ␤-lacta...

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Section: Role Of Lys 392 As the General Base In Acylation-mentioning

confidence: 81%

Section: Role Of Lys 392 As the General Base In Acylation-mentioning

confidence: 99%

See 1 more Smart Citation

Crystal Structures of the Apo and Penicillin-acylated Forms of the BlaR1 β-Lactam Sensor of Staphylococcus aureus

Wilke

Hills

Zhang

et al. 2004

Journal of Biological Chemistry

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“…This arrangement of Ser-X-X-Lys for PBPs and related ␤-lactamases is understood to be important for the mechanisms of these enzymes (30). We have shown that when the corresponding lysine is mutated to alanine in the OXA-10 ␤-lactamase, the enzyme cannot undergo acylation by its substrate (31). A similar mutation in the penicillin-binding protein BlaR from S. aureus was shown to attenuate the rate of protein acylation by 6730-fold (32).…”

Section: Resultsmentioning

confidence: 99%

The Basis for Resistance to β-Lactam Antibiotics by Penicillin-binding Protein 2a of Methicillin-resistant Staphylococcus aureus

Fuda

Suvorov

Vakulenko

et al. 2004

Journal of Biological Chemistry

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221

200

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Penicillin-binding protein 2a (PBP2a) of Staphylococcus aureus is refractory to inhibition by available ␤-lactam antibiotics, resulting in resistance to these antibiotics. The strains of S. aureus that have acquired the mecA gene for PBP2a are designated as methicillin-resistant S. aureus (MRSA). The mecA gene was cloned and expressed in Escherichia coli, and PBP2a was purified to homogeneity. The kinetic parameters for interactions of several ␤-lactam antibiotics (penicillins, cephalosporins, and a carbapenem) and PBP2a were evaluated. The enzyme manifests resistance to covalent modification by ␤-lactam antibiotics at the active site serine residue in two ways. First, the microscopic rate constant for acylation (k 2 ) is attenuated by 3 to 4 orders of magnitude over the corresponding determinations for penicillinsensitive penicillin-binding proteins. Second, the enzyme shows elevated dissociation constants (K d ) for the non-covalent pre-acylation complexes with the antibiotics, the formation of which ultimately would lead to enzyme acylation. The two factors working in concert effectively prevent enzyme acylation by the antibiotics in vivo, giving rise to drug resistance. Given the opportunity to form the acyl enzyme species in in vitro experiments, circular dichroism measurements revealed that the enzyme undergoes substantial conformational changes in the course of the process that would lead to enzyme acylation. The observed conformational changes are likely to be a hallmark for how this enzyme carries out its catalytic function in cross-linking the bacterial cell wall.

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“…APH(2 00 )-IVa operates via a sequential mechanism in which the two substrates (NTP and aminoglycoside) bind in a random manner. All aminoglycoside-modifying enzymes studied to date (APH(3 0 )-Ia, APH(3 0 )-IIa, APH(2 00 )-Ia, APH(2 00 )-IIa, and APH(2 00 )-IIIa) operate via the sequential mechanism, 21,23,[26][27][28][29][30][31][32][33] and use a random mechanism for turnover chemistry, 21,23,31,33,34 with the exception of APH(3 0 )-IIIa, which has been shown to use a Theorell-Chance mechanism with compulsory ordered substrate binding and product release. 35 …”

Section: Kinetic Mechanism Of Aph(2 00 )-Ivamentioning

confidence: 99%

Crystal structure and kinetic mechanism of aminoglycoside phosphotransferase‐2″‐IVa

et al. 2010

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Acquired resistance to aminoglycoside antibiotics primarily results from deactivation by three families of aminoglycoside-modifying enzymes. Here, we report the kinetic mechanism and structure of the aminoglycoside phosphotransferase 2 00 -IVa (APH(2 00 )-IVa), an enzyme responsible for resistance to aminoglycoside antibiotics in clinical enterococcal and staphylococcal isolates. The enzyme operates via a Bi-Bi sequential mechanism in which the two substrates (ATP or GTP and an aminoglycoside) bind in a random manner. The APH(2 00 )-IVa enzyme phosphorylates various 4,6-disubstituted aminoglycoside antibiotics with catalytic efficiencies (k cat /K m ) of 1.5 3 10 3 to 1.2 3 10 6 (M 21 s 21 ). The enzyme uses both ATP and GTP as the phosphate source, an extremely rare occurrence in the phosphotransferase and protein kinase enzymes. Based on an analysis of the APH(2 00 )-IVa structure, two overlapping binding templates specifically tuned for hydrogen bonding to either ATP or GTP have been identified and described. A detailed understanding of the structure and mechanism of the GTP-utilizing phosphotransferases is crucial for the development of either novel aminoglycosides or, more importantly, GTP-based enzyme inhibitors which would not be expected to interfere with crucial ATP-dependent enzymes.

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Critical involvement of a carbamylated lysine in catalytic function of class D β-lactamases

Cited by 220 publications

References 24 publications

Crystal Structures of the Apo and Penicillin-acylated Forms of the BlaR1 β-Lactam Sensor of Staphylococcus aureus

Crystal Structures of the Apo and Penicillin-acylated Forms of the BlaR1 β-Lactam Sensor of Staphylococcus aureus

The Basis for Resistance to β-Lactam Antibiotics by Penicillin-binding Protein 2a of Methicillin-resistant Staphylococcus aureus

Crystal structure and kinetic mechanism of aminoglycoside phosphotransferase‐2″‐IVa

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