Structure of the wild-type TEM-1 β-lactamase at 1.55 Å and the mutant enzyme Ser70Ala at 2.1 Å suggest the mode of noncovalent catalysis for the mutant enzyme

Stec, Boguslaw; Holtz, Kathleen M.; Wojciechowski, Cheryl L.; Kantrowitz, Evan R.

doi:10.1107/s0907444905014356

Cited by 69 publications

(93 citation statements)

References 30 publications

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“…The presence of the omega-loop-like structure in the sMSMEG_2433 model might drive the side chain of Glu75 into the active site, thereby bringing water molecules between the Glu75 and Lys48. It is important to mention that the deacylation of acyl enzyme intermediate involves the formation of hydrogen bridges bound with an activated water molecule to Glu75, releasing Ser45 from the betalactam ring in its protonated form (Stec et al, 2005). Structural analysis of the sMSMEG_2433 model predicts a high deacylation rate owing to the presence of the omega loop, which is in agreement with the high value of the deacylation rate constant of the enzyme obtained kinetically.…”

Section: Discussion Smsmeg_2433 Exhibits Higher Dd-cpase Activity Witsupporting

confidence: 67%

“…sMSMEG_2433 shows a slow acylation rate but significant deacylation rate towards penicillin Generally, the beta-lactam hydrolysis mechanism is mediated by two principal processes, acylation and deacylation (Stec et al, 2005). In acylation, the serine residue of SXXK is deprotonated to execute a nucleophilic attack at the carbon atom in the carbonyl group of the beta-lactam ring, and active site residues form a conducive environment for hydrogen bond formation around their respective amide bonds (Massova & Mobashery, 1998;Vakulenko et al, 1999).…”

Section: Discussion Smsmeg_2433 Exhibits Higher Dd-cpase Activity Witmentioning

confidence: 99%

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A putative low-molecular-mass penicillin-binding protein (PBP) of Mycobacterium smegmatis exhibits prominent physiological characteristics of dd-carboxypeptidase and beta-lactamase

et al. 2015

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DD-Carboxypeptidases (DD-CPases) are low-molecular-mass (LMM) penicillin-binding proteins (PBPs) that are mainly involved in peptidoglycan remodelling, but little is known about the DD-CPases of mycobacteria. In this study, a putative DD-CPase of Mycobacterium smegmatis, MSMEG_2433 is characterized. The gene for the membrane-bound form of MSMEG_2433 was cloned and expressed in Escherichia coli in its active form, as revealed by its ability to bind to the Bocillin-FL (fluorescent penicillin). Interestingly, in vivo expression of MSMEG_2433 could restore the cell shape oddities of the septuple PBP mutant of E. coli, which was a prominent physiological characteristic of DD-CPases. Moreover, expression of MSMEG_2433 in trans elevated beta-lactam resistance in PBP deletion mutants (DdacAdacC) of E. coli, strengthening its physiology as a DD-CPase. To confirm the biochemical reason behind such physiological behaviours, a soluble form of MSMEG_2433 (sMSMEG_2433) was created, expressed and purified. In agreement with the observed physiological phenomena, sMSMEG_2433 exhibited DD-CPase activity against artificial and peptidoglycan-mimetic DD-CPase substrates. To our surprise, enzymic analyses of MSMEG_2433 revealed efficient deacylation for beta-lactam substrates at physiological pH, which is a unique characteristic of beta-lactamases. In addition to the MSMEG_2433 active site that favours DD-CPase activity, in silico analyses also predicted the presence of an omega-loop-like region in MSMEG_2433, which is an important determinant of its beta-lactamase activity. Based on the in vitro, in vivo and in silico studies, we conclude that MSMEG_2433 is a dual enzyme, possessing both DD-CPase and beta-lactamase activities.

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Section: Discussion Smsmeg_2433 Exhibits Higher Dd-cpase Activity Witsupporting

confidence: 67%

Section: Discussion Smsmeg_2433 Exhibits Higher Dd-cpase Activity Witmentioning

confidence: 99%

A putative low-molecular-mass penicillin-binding protein (PBP) of Mycobacterium smegmatis exhibits prominent physiological characteristics of dd-carboxypeptidase and beta-lactamase

et al. 2015

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“…In KPC-2, alteration of four residues (Ser70, Ser130, Asn132, and Asn170) of carbapenemases (KPC-2 [38], NMC-A [40], and SME-1 [39]) compared with non-carbapenemase (SHV-1 [60] and TEM-1 [61]) has been observed [38]. The shift of key residues such as Ser70, Ser130, Asn132, and Asn170 in SFC-1 compared with non-carbapenemases (SHV-1 [60], CTX-M-16 [62], BlaC [63], and TEM-1 [61]) has also been revealed [37].…”

Section: Non-metallo-carbapenemases: Zinc-independent Classes a Cmentioning

confidence: 99%

“…The shift of key residues such as Ser70, Ser130, Asn132, and Asn170 in SFC-1 compared with non-carbapenemases (SHV-1 [60], CTX-M-16 [62], BlaC [63], and TEM-1 [61]) has also been revealed [37]. A less buried position of side chain of Ser70 in the active site of carbapenemases allows easier access of the bulkier substrates (Figure 5).…”

Section: Non-metallo-carbapenemases: Zinc-independent Classes a Cmentioning

confidence: 99%

Structural Basis for Carbapenem-Hydrolyzing Mechanisms of Carbapenemases Conferring Antibiotic Resistance

Jeon

Lee

et al. 2015

IJMS

159

121

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Carbapenems (imipenem, meropenem, biapenem, ertapenem, and doripenem) are β-lactam antimicrobial agents. Because carbapenems have the broadest spectra among all β-lactams and are primarily used to treat infections by multi-resistant Gram-negative bacteria, the emergence and spread of carbapenemases became a major public health concern. Carbapenemases are the most versatile family of β-lactamases that are able to hydrolyze carbapenems and many other β-lactams. According to the dependency of divalent cations for enzyme activation, carbapenemases can be divided into metallo-carbapenemases (zinc-dependent class B) and non-metallo-carbapenemases (zinc-independent classes A, C, and D). Many studies have provided various carbapenemase structures. Here we present a comprehensive and systematic review of three-dimensional structures of carbapenemase-carbapenem complexes as well as those of carbapenemases. We update recent studies in understanding the enzymatic mechanism of each class of carbapenemase, and summarize structural insights about regions and residues that are important in acquiring the carbapenemase activity.

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“…The class C enzymes include P99, a chromosome-encoded cephalosporinase from Enterobacter cloacae P99 (26) that was identified as a clavulanate-resistant enzyme more than 30 years ago (31). The structural and enzymological properties of both enzymes and their naturally occurring and laboratory-generated mutant variants have been intensively studied for many years, and thus they present as model enzymes for investigation of inhibition by novel compounds (8,17,24,37,38).…”

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confidence: 99%

Mechanistic Studies of the Inactivation of TEM-1 and P99 by NXL104, a Novel Non-β-Lactam β-Lactamase Inhibitor

Stachyra¹,

Péchereau²,

Bruneau³

et al. 2010

Antimicrob Agents Chemother

134

125

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NXL104 is a potent inhibitor of class A and C serine ␤-lactamases, including KPC carbapenemases. Native and NXL104-inhibited TEM-1 and P99 ␤-lactamases analyzed by liquid chromatography-electrospray ionization-time of flight mass spectrometry revealed that the inactivated enzymes formed a covalent adduct with NXL104. The principal inhibitory characteristics of NXL104 against TEM-1 and P99 ␤-lactamases were determined, including partition ratios, dissociation constants (K), rate constants for deactivation (k 2 ), and reactivation rates. NXL104 is a potent inhibitor of TEM-1 and P99, characterized by high carbamylation efficiencies (k 2 /K of 3.7 ؋ 10 5 M ؊1 s ؊1 for TEM-1 and 1 ؋ 10 4 M ؊1 s ؊1 for P99) and slow decarbamylation. Complete loss of ␤-lactamase activity was obtained at a 1/1 enzyme/NXL104 ratio, with a k 3 value (rate constant for formation of product and free enzyme) close to zero for TEM-1 and P99. Fifty percent inhibitory concentrations (IC 50 s) were evaluated on selected ␤-lactamases, and NXL104 was shown to be a very potent inhibitor of class A and C ␤-lactamases. IC 50 s obtained with NXL104 (from 3 nM to 170 nM) were globally comparable on the ␤-lactamases CTX-M-15 and SHV-4 with those obtained with the comparators (clavulanate, tazobactam, and sulbactam) but were far lower on TEM-1, KPC-2, P99, and AmpC than those of the comparators. In-depth studies on TEM-1 and P99 demonstrated that NXL104 had a comparable or better affinity and inactivation rate than clavulanate and tazobactam and in all cases an improved stability of the covalent enzyme/inhibitor complex.

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Structure of the wild-type TEM-1 β-lactamase at 1.55 Å and the mutant enzyme Ser70Ala at 2.1 Å suggest the mode of noncovalent catalysis for the mutant enzyme

Cited by 69 publications

References 30 publications

A putative low-molecular-mass penicillin-binding protein (PBP) of Mycobacterium smegmatis exhibits prominent physiological characteristics of dd-carboxypeptidase and beta-lactamase

A putative low-molecular-mass penicillin-binding protein (PBP) of Mycobacterium smegmatis exhibits prominent physiological characteristics of dd-carboxypeptidase and beta-lactamase

Structural Basis for Carbapenem-Hydrolyzing Mechanisms of Carbapenemases Conferring Antibiotic Resistance

Mechanistic Studies of the Inactivation of TEM-1 and P99 by NXL104, a Novel Non-β-Lactam β-Lactamase Inhibitor

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