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...
The TEM-1 β-lactamase confers bacterial resistance to penicillin antibiotics and has acquired mutations that permit the enzyme to hydrolyze extended spectrum cephalosporins or avoid inactivation by β-lactamase inhibitors. However, many of these substitutions have been shown to reduce activity against penicillin antibiotics and/or result in a loss of stability for the enzyme. In order to gain more information concerning the trade-offs associated with active site substitutions, a genetic selection was used to find second site mutations which partially restore ampicillin resistance levels conferred by an R244A active site TEM-1 β-lactamase mutant. An L201P substitution distant from the active site was identified that enhanced ampicillin resistance levels and increased protein expression levels of the R244A TEM-1 mutant. The L201P substitution also increases the ampicillin resistance levels and restores expression levels of a poorly expressed TEM-1 mutant with a coredisrupting substitution. In vitro thermal denaturation of purified protein indicated that the L201P mutation increases the T m of the TEM-1 enzyme. The X-ray structure of the L201P TEM-1 mutant was determined to gain insight into the increase in enzyme stability. The proline substitution occurs at the N-terminus of an α-helix and may stabilize the enzyme by reducing the helix dipole as well as lowering the conformational entropy cost of folding due to the reduced number of conformations available in the unfolded state. Collectively the data suggest that L201P promotes tolerance of some deleterious TEM-1 mutations by enhancing protein stability of these mutants.
Pre-organization of enzyme active sites for substrate recognition typically comes at a cost to the stability of the folded form of the protein, and consequently enzymes can be dramatically stabilized by substitutions that attenuate the size and pre-organization “strain” of the active site. How this stability-activity trade-off constrains enzyme evolution has remained less certain, and it is unclear whether one should expect major stability insults as enzymes mutate towards new activities, or how these new activities manifest structurally. These questions are both germane and easy to study in β-lactamases, which are evolving on the timescale of years to confer resistance to an ever-broader spectrum of β-lactam antibiotics. To explore whether stability is a substantial constraint on this antibiotic resistance evolution, we investigated extended-spectrum mutants of class C β-lactamases which had evolved new activity versus third-generation cephalosporins. Five mutant enzymes had between 100- to 200-fold increased activity against the antibiotic cefotaxime in enzyme assays, and the mutant enzymes all lost thermodynamic stability – from 1.7 to 4.1 kcal/mol – consistent with the function-stability hypothesis. Intriguingly, several of the substitutions were 10 – 20 Å from the catalytic serine; the question arose how they conferred extended-spectrum activity. Eight structures, including complexes with inhibitors and extended-spectrum antibiotics, were determined by x-ray crystallography. Distinct mechanisms of action are revealed for each mutant, including changes in the flexibility and ground state structures of the enzyme. These results explain the structural bases for the antibiotic resistance conferred by these substitutions, and their corresponding decrease in protein stability, which will constrain the evolution of new antibiotic resistance.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.