Structure of the SHV-1 β-Lactamase

Kuzin, A.P.; Nukaga, Michiyoshi; Nukaga, Yasuko; Hujer, Andrea M.; Bonomo, Robert A.; Knox, James R.

doi:10.1021/bi990136d

Cited by 116 publications

(124 citation statements)

References 40 publications

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“…One explanation for the catalytic deficit in TEM mutants at position 244 is displacement of that water molecule, which is essential for secondary clavulanate ring opening in the inactivation pathway. Comparing the apo-enzyme crystal structures of TEM-1 (26) and SHV-1 (25), the distances between the closest guanidinium nitrogen of Arg-244 and the backbone carbonyl oxygen Val-216 are significantly different (Fig. 7).…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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

Self Cite

109

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“…The structure of the S130G mutant was recently determined to 1.8 Å in SHV β-lactamase (35), which has 68% sequence identity to TEM-1 and is closely related structurally; their structures have a C α RMSD of 1.4 Å, and the catalytically important residues have an RMSD of only 0.23 Å (36,37). Despite these similarities, the Ser130Gly substitution in TEM leads to related, but nevertheless different, conformational changes compared to SHV S130G.…”

Section: Discussionmentioning

confidence: 99%

Structural Consequences of the Inhibitor-Resistant Ser130Gly Substitution in TEM β-Lactamase^,

et al. 2005

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Abstractβ-Lactamase confers resistance to penicillin-like antibiotics by hydrolyzing their β-lactam bond. To combat these enzymes, inhibitors covalently cross-linking the hydrolytic Ser70 to Ser130 were introduced. In turn, mutant β-lactamases have emerged with decreased susceptibility to these mechanism-based inhibitors. Substituting Ser130 with glycine in the inhibitor-resistant TEM (IRT) mutant TEM-76 (S130G) prevents the irreversible cross-linking step. Since the completely conserved Ser130 is thought to transfer a proton important for catalysis, its substitution might be hypothesized to result in a nonfunctional enzyme; this is clearly not the case. To investigate how TEM-76 remains active, its structure was determined by X-ray crystallography to 1.40 Å resolution. A new water molecule (Wat1023) is observed in the active site, with two configurations located 1.1 and 1.3 Å from the missing Ser130 Oγ; this water molecule likely replaces the Ser130 side-chain hydroxyl in substrate hydrolysis. Intriguingly, this same water molecule is seen in the IRT TEM-32 (M69I/ M182T), where Ser130 has moved significantly. TEM-76 shares other structural similarities with various IRTs; like TEM-30 (R244S) and TEM-84 (N276D), the water molecule activating clavulanate for cross-linking (Wat1614) is disordered (in TEM-30 it is actually absent). As expected, TEM-76 has decreased kinetic activity, likely due to the replacement of the Ser130 side-chain hydroxyl with a water molecule. In contrast to the recently determined structure of the S130G mutant in the related SHV-1 β-lactamase, in TEM-76 the key hydrolytic water (Wat1561) is still present. The conservation of similar accommodations among IRT mutants suggests that resistance arises from common mechanisms, despite the disparate locations of the various substitutions.The catalytic activity of the TEM family of class A β-lactamases is a major resistance mechanism against β-lactam antibiotics, such as penicillins ( Figure 1A); hydrolysis of the β-lactam ring of these antibiotics renders them ineffective against bacteria. To combat these enzymes, inhibitors against β-lactamase, such as clavulanate, tazobactam, and sulbactam ( Figure 1B-D), were developed. In turn, inhibitor-resistant TEM β-lactamases (IRTs) 1 have emerged in the clinic with decreased susceptibility to these mechanism-based inhibitors. These † This work was supported by NIH Grants GM63815 (to B.K.S.) and AI33170 (to S.M.) and by the Achievement Rewards for College Scientists Foundation and a National Science Foundation predoctoral fellowship (to V.L.T.). ‡ Atomic coordinates and structure factors have been deposited in the Protein Data Bank at the Research Collaboratory for Structural Bioinformatics at Rutgers University (entry 1YT4). * Corresponding authors. B.K.S.: phone, 415-514-4126; fax, 415-502-1411; e-mail,shoichet@cgl.ucsf.edu. S.M.: phone, 574-631-2933; fax, 574-631-6652; e-mail,mobashery@nd.edu inhibitors function by acylating the enzyme's active site. After reacting with the catalytic Ser70, these β-lac...

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“…Molecular Modeling-Insight II™ software (Accelrys) was used to construct the S130G variant and to perform energy minimizations as described previously (9) based on the SHV-1 crystal structure (Protein Data Bank code 1SHV) (21).…”

Section: Mutagenesis Of Ser-130 In Shv ␤-Lactamasementioning

confidence: 99%

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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Structure of the SHV-1 β-Lactamase

Cited by 116 publications

References 40 publications

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

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

Structural Consequences of the Inhibitor-Resistant Ser130Gly Substitution in TEM β-Lactamase^,

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

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Structure of the SHV-1 β-Lactamase

Cited by 116 publications

References 40 publications

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

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

Structural Consequences of the Inhibitor-Resistant Ser130Gly Substitution in TEM β-Lactamase,

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

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Structural Consequences of the Inhibitor-Resistant Ser130Gly Substitution in TEM β-Lactamase^,