Amino Acid Substitutions at Ambler Position Gly238 in the SHV-1 β-Lactamase: Exploring Sequence Requirements for Resistance to Penicillins and Cephalosporins

Hujer, Andrea M.; Hujer, Kristine M.; Helfand, Marion S.; Anderson, Vernon E.

doi:10.1128/aac.46.12.3971-3977.2002

Cited by 35 publications

(58 citation statements)

References 30 publications

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“…Express automated DNA sequencer (Amersham Biosciences) using the Thermo Sequenase TM fluorescent-labeled primer cycle sequencing kit in a manner similar to methods published previously (8,9,15).…”

Section: Methodsmentioning

confidence: 99%

“…Antibiotics used and their suppliers were described previously (8,9,15). Concentrations employed for determining MIC values were in g/ml.…”

Section: Methodsmentioning

confidence: 99%

“…Mutagenesis-PCR-based site-saturation and site-directed mutagenesis was performed using a QuikChange™ mutagenesis kit (Stratagene), as reported (9,15). Two complementary degenerate oligonucleotides (Genosys Biotechnologies, The Woodlands, TX) were first constructed for site-saturation mutagenesis at the Ser-130 position.…”

Section: Methodsmentioning

confidence: 99%

“…␤-Lactamase Purification-SHV-1, S130G, and S130T ␤-lactamases were purified to homogeneity from E. coli DH10B cells according to a method employing preparative isoelectric focusing (8,9,15). We assessed the purity of each ␤-lactamase on 5% stacking and 12% resolving SDS-PAGE gels.…”

Section: Determination Of Steady-state Expression Using An Enzyme-linkedmentioning

confidence: 99%

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Understanding Resistance to β-Lactams and β-Lactamase Inhibitors in the SHV β-Lactamase

Helfand

Bethel

Hujer

et al. 2003

Journal of Biological Chemistry

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Bacterial resistance to ␤-lactam/␤-lactamase inhibitor combinations by single amino acid mutations in class A ␤-lactamases threatens our most potent clinical antibiotics. In TEM-1 and SHV-1, the common class A ␤-lactamases, alterations at Ser-130 confer resistance to inactivation by the ␤-lactamase inhibitors, clavulanic acid, and tazobactam. By using site-saturation mutagenesis, we sought to determine the amino acid substitutions at Ser-130 in SHV-1 ␤-lactamase that result in resistance to these inhibitors. Antibiotic susceptibility testing revealed that ampicillin and ampicillin/clavulanic acid resistance was observed only for the S130G ␤-lactamase expressed in Escherichia coli. Kinetic analysis of the S130G ␤-lactamase demonstrated a significant elevation in apparent K m and a reduction in k cat /K m for ampicillin. Marked increases in the dissociation constant for the preacylation complex, K I , of clavulanic acid (SHV-1, 0.14 M; S130G, 46.5 M) and tazobactam (SHV-1, 0.07 M; S130G, 4.2 M) were observed. In contrast, the k inact s of S130G and SHV-1 differed by only 17% for clavulanic acid and 40% for tazobactam. Progressive inactivation studies showed that the inhibitor to enzyme ratios required to inactivate SHV-1 and S130G were similar. Our observations demonstrate that enzymatic activity is preserved despite amino acid substitutions that significantly alter the apparent affinity of the active site for ␤-lactams and ␤-lactamase inhibitors. These results underscore the mechanistic versatility of class A ␤-lactamases and have implications for the design of novel ␤-lactamase inhibitors.

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Section: Methodsmentioning

confidence: 99%

“…Antibiotics used and their suppliers were described previously (8,9,15). Concentrations employed for determining MIC values were in g/ml.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Determination Of Steady-state Expression Using An Enzyme-linkedmentioning

confidence: 99%

See 2 more Smart Citations

Understanding Resistance to β-Lactams and β-Lactamase Inhibitors in the SHV β-Lactamase

Helfand

Bethel

Hujer

et al. 2003

Journal of Biological Chemistry

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“…Because the R244K, -Met, and -Phe mutants were not obtained in the initial screen, bla SHV-1(R244K) , bla , and bla SHV-1(R244F) were constructed by site-directed mutagenesis, using specific mutagenic oligonucleotides as previously described (18). DNA sequencing confirmed the presence of the mutated codons as described above.…”

Section: Methodsmentioning

confidence: 99%

Probing Active Site Chemistry in SHV β-Lactamase Variants at Ambler Position 244

Thomson

Distler

Prati

et al. 2006

Journal of Biological Chemistry

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109

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Inhibitor-resistant class A ␤-lactamases are an emerging threat to the use of ␤-lactam/␤-lactamase inhibitor combinations (e.g. amoxicillin/clavulanate) in the treatment of serious bacterial infections. In the TEM family of Class A ␤-lactamases, single amino acid substitutions at Arg-244 confer resistance to clavulanate inactivation. To understand the amino acid sequence requirements in class A ␤-lactamases that confer resistance to clavulanate, we performed site-saturation mutagenesis of Arg-244 in SHV-1, a related class A ␤-lactamase found in Klebsiella pneumoniae. Twelve SHV enzymes with amino acid substitutions at Arg-244 resulted in significant increases in minimal inhibitory concentrations to ampicillin/ clavulanate when expressed in Escherichia coli. Kinetic analyses of SHV-1, R244S, R244Q, R244L, and R244E ␤-lactamases revealed that the main determinant of clavulanate resistance was reduced inhibitor affinity. In contrast to studies in the highly similar TEM enzyme, we observed increases in clavulanate k inact for all mutants. Electrospray ionization mass spectrometry of clavulanate inhibited SHV-1 and R244S showed nearly identical mass adducts, arguing against a difference in the inactivation mechanism. Testing a wide range of substrates with C 3-4 carboxylates in different stereochemical orientations, we observed impaired affinity for all substrates among inhibitor resistant variants. Lastly, we synthesized two boronic acid transition state analogs that mimic cephalothin and found substitutions at Arg-244 markedly affect both the affinity and kinetics of binding to the chiral, deacylation transition state inhibitor. These data define a role for Arg-244 in substrate and inhibitor binding in the SHV ␤-lactamase.␤-Lactams have been the cornerstone of antibacterial chemotherapy since the introduction of penicillin. Although this family of antibiotics now contains numerous classes (penicillins, cephalosporins, and carbapenems, see Fig. 1), their use is threatened by the expansion and spread of ␤-lactamase enzymes (1). These bacterial enzymes hydrolyze ␤-lactam antibiotics before reaching their target, the penicillin-binding proteins. In an effort to retain the utility of several generations of life-saving ␤-lactam antibiotics, ␤-lactamase inhibitors (clavulanate, sulbactam, and tazobactam) have been developed. These mechanism-based inhibitors are ␤-lactam compounds that experience multistep reactions within the active site of ␤-lactamase enzymes (2-4). Typically the inhibitors lack antimicrobial activity and are formulated with a ␤-lactam antibiotic to act as shields against ␤-lactamase enzymes. This allows the ␤-lactam to bypass the ␤-lactamase and inactivate the penicillin binding proteins.Unfortunately, inhibitor-resistant class A ␤-lactamases are emerging in the clinic and undermining the use of ␤-lactam/␤-lactamase inhibitor therapy (5-7). Interest has been renewed in the discovery of novel ␤-lactamase inactivators to circumvent these inhibitor-resistant enzymes (8 -11). In this context, detailed analy...

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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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Amino Acid Substitutions at Ambler Position Gly238 in the SHV-1 β-Lactamase: Exploring Sequence Requirements for Resistance to Penicillins and Cephalosporins

Cited by 35 publications

References 30 publications

Understanding Resistance to β-Lactams and β-Lactamase Inhibitors in the SHV β-Lactamase

Understanding Resistance to β-Lactams and β-Lactamase Inhibitors in the SHV β-Lactamase

Probing Active Site Chemistry in SHV β-Lactamase Variants at Ambler Position 244

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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