Overexpression and Biosynthetic Deuterium Enrichment of TEM-1 β-Lactamase for Structural Characterization by Magnetic Resonance Methods

Sosa‐Peinado, Alejandro; Mustafi, Devkumar; Makinen, Marvin W.

doi:10.1006/prep.2000.1243

Cited by 37 publications

(34 citation statements)

References 49 publications

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“…A pET-24 vector containing the sequence encoding TEM-1 fused to the leader sequence of OmpA was used to overexpress the enzyme (35). TEM-1 was purified from the culture supernatant (33).…”

Section: Methodsmentioning

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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“…A pET-24 vector containing the sequence encoding TEM-1 fused to the leader sequence of OmpA was used to overexpress the enzyme (35). TEM-1 was purified from the culture supernatant (33).…”

Section: Methodsmentioning

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

View full text Add to dashboard Cite

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“…The pET-TEM-1 vector encodes an ompA leader-TEM-1 fusion gene driven by the T7 promoter. 35 The TEM-1 enzyme encoded in pET-TEM-1 contains a noncanonical Glu28Gly substitution as a result of a g!a nucleotide mutation present in the parent vector. All b-lactamases used in this study are derived from the pET-TEM-1 plasmid and therefore contain the Glu28Gly substitution.…”

Section: Methodsmentioning

confidence: 99%

Analysis of the plasticity of location of the Arg244 positive charge within the active site of the TEM‐1 β‐lactamase

2009

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A large number of b-lactamases have emerged that are capable of conferring bacterial resistance to b-lactam antibiotics. Comparison of the structural and functional features of this family has refined understanding of the catalytic properties of these enzymes. An arginine residue present at position 244 in TEM-1 b-lactamase interacts with the carboxyl group common to penicillin and cephalosporin antibiotics and thereby stabilizes both the substrate and transition state complexes. A comparison of class A b-lactamase sequences reveals that arginine at position 244 is not conserved, although a positive charge at this structural location is conserved and is provided by an arginine at positions 220 or 276 for those enzymes lacking arginine at position 244. The plasticity of the location of positive charge in the b-lactamase active site was experimentally investigated by relocating the arginine at position 244 in TEM-1 b-lactamase to positions 220, 272, and 276 by site-directed mutagenesis. Kinetic analysis of the engineered b-lactamases revealed that removal of arginine 244 by alanine mutation reduced catalytic efficiency against all substrates tested and restoration of an arginine at positions 272 or 276 partially suppresses the catalytic defect of the Arg244Ala substitution. These results suggest an evolutionary mechanism for the observed divergence of the position of positive charge in the active site of class A b-lactamases.

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“…However, the ␤-lactamases were poorly expressed in the original constructs. In this work, a high-level expression system was modified to overexpress these enzymes as ompA-bla1 and ompA-bla2 gene fusion products (25). The gene for mature Bla1 or Bla2 enzyme was fused to the leader sequence of the E. coli OmpA protein, cloned into pET24a(ϩ), and transformed into E. coli BL21Star(DE3).…”

mentioning

confidence: 99%

“…Overlap extension PCR was employed as previously described (12) to construct pCMOB1 and pCMOB2. Briefly, four primers, ompA-bla1-bot, ompA-bla1-top, bla1-BamHI, and pET-SphI (Table 1), were used to PCR amplify two DNA fragments with overlapping ends from vectors pET24a(ϩ)-TEM1 (containing the ompA signal sequence) and pUTE523 (containing bla1) (25). The two overlapping PCR products were combined in a single PCR and amplified by using the external primers bla1-BamHI and pET-SphI to generate the fusion product, ompA-bla1.…”

mentioning

confidence: 99%

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Biochemical Characterization of β-Lactamases Bla1 and Bla2 from Bacillus anthracis

Materon

Queenan

Koehler³

et al. 2003

Antimicrob Agents Chemother

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The Sterne and Ames strains of Bacillus anthracis carry chromosomal genes bla1 and bla2, which confer ␤-lactam resistance when expressed in Escherichia coli. MIC measurements and steady-state kinetic analyses indicate that Bla1 possesses penicillinase activity while Bla2 possesses penicillinase, cephalosporinase, and carbapenem-hydrolyzing activities.Anthrax, caused by Bacillus anthracis, can be treated with antibiotics such as tetracyclines, macrolides, quinolones, and ␤-lactams. However, recent studies have shown that naturally occurring B. anthracis isolates can show variable sensitivity to multiple classes of antibiotics (5,7,20,23 (1,14), present in group 2 ␤-lactamases. Bla2 shares 92% amino acid identity with the group 3 B. cereus 569H enzyme (17), shows conservation of the zinc-chelating motif common to group 3 enzymes (15), and is EDTA susceptible (data not shown).The bla1 and bla2 genes were previously cloned from the B. anthracis Sterne strain and expressed in Escherichia coli (8). However, the ␤-lactamases were poorly expressed in the original constructs. In this work, a high-level expression system was modified to overexpress these enzymes as ompA-bla1 and ompA-bla2 gene fusion products (25). The gene for mature Bla1 or Bla2 enzyme was fused to the leader sequence of the E. coli OmpA protein, cloned into pET24a(ϩ), and transformed into E. coli BL21Star(DE3). pET24a(ϩ) contains an IPTG (isopropyl-␤-D-thiogalactopyranoside)-inducible T7lacUV5 promoter for large-scale protein expression and a Kan r marker. Overlap extension PCR was employed as previously described (12) to construct pCMOB1 and pCMOB2. Briefly, four primers, ompA-bla1-bot, ompA-bla1-top, bla1-BamHI, and pET-SphI (Table 1), were used to PCR amplify two DNA fragments with overlapping ends from vectors pET24a(ϩ)-TEM1 (containing the ompA signal sequence) and pUTE523 (containing bla1) (25). The two overlapping PCR products were combined in a single PCR and amplified by using the external primers bla1-BamHI and pET-SphI to generate the fusion product, ompA-bla1. The fusion product was digested with BamHI and XbaI and ligated into pET-24a(ϩ). Ligation products were transformed into E. coli BL21Star (DE3), and plasmid DNA was isolated from a set of transformants. The sequence of the ompA-bla1 fusion gene was determined and confirmed by ABI3100 automated sequencing. The same procedure was used to construct pCMOB2. The four primers, ompA-bla2-bot, ompA-bla2-top, bla2-BamHI, and pETSphI (Table 1), were used to PCR amplify two DNA fragments with overlapping ends from vectors pET24a(ϩ)-TEM1 and pUTE490 (containing bla2) (25).The MICs of ␤-lactam antibiotics were determined by using twofold dilutions in Luria-Bertani-kanamycin broth (LB-Kan; 25 g/ml) ( Table 2). Kanamycin was added to maintain the plasmid. In the absence of IPTG, expression of the cloned genes still occurs; therefore, IPTG was not added to the LBKan broth. An inoculum of 10 5 E. coli BL21Star(DE3) cells/ml containing pCMOB1, pCMOB2, or the pET24a(ϩ) religated vector was used. The MIC...

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Overexpression and Biosynthetic Deuterium Enrichment of TEM-1 β-Lactamase for Structural Characterization by Magnetic Resonance Methods

Cited by 37 publications

References 49 publications

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

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

Analysis of the plasticity of location of the Arg244 positive charge within the active site of the TEM‐1 β‐lactamase

Biochemical Characterization of β-Lactamases Bla1 and Bla2 from Bacillus anthracis

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