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

Abstract

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...

N152G, -S, and -T Substitutions in CMY-2 β-Lactamase Increase Catalytic Efficiency for Cefoxitin and Inactivation Rates for Tazobactam

“…Lefurgy et al examined the kinetic properties of N152S and -T P99 variants with penicillin and cephalosporin substrates and concluded that hydrogen bonding of S152 or T152 to K67 or to the substrate accounted for the preservation of catalytic activity (16). They hypothesized that a water molecule can play a catalytic role in the N152G variant of P99, as has been shown for S130G SHV (25,37,38). The recently reported crystal structure of the N152G P99 variant shows considerable space left by the N152G mutation, occupied by the His 6 tag at the C terminus of the protein (39).…”

Can Inhibitor-Resistant Substitutions in the Mycobacterium tuberculosis β-Lactamase BlaC Lead to Clavulanate Resistance?: a Biochemical Rationale for the Use of β-Lactam–β-Lactamase Inhibitor Combinations

“…The latter step seems to be important for permanent inactivation of certain ␤-lactamases. Indeed, several inhibitor-resistant class A variant enzymes which bear a serine-to-glycine mutation have been described (60)(61)(62).…”

Three Decades of β-Lactamase Inhibitors

“…Amino acid substitutions in TEM and SHV result in resistance to clavulanate by two primary mechanisms: (i) alterations in the geometry of shape of the oxyanion hole and (ii) changes in the position of Ser130 or Arg244 (33,73,101,120,408,411,416). The individual residues that are most commonly substituted in IR TEM enzymes are Arg244, the nearby Asn276 and Arg275, Met69, and the active-site Ser130 (www .lahey.org/studies/webt.asp) (Fig.…”

“…Removing the cross-linking residue seems a clear way to overcome inactivation, but inhibitors, and particularly tazobactam, retain efficacy against the Ser130Gly enzyme. Based on these mechanistic roles, how does the Ser130Gly variant maintain hydrolytic activity and why does it confer resistance to inhibitors (408)? In response to the first part of the question, evidence from X-ray crystallography of SHV and TEM Ser130Gly (PDB 1TDL and 1YT4, respectively) confirmed that a relocated water molecule, initially predicted by Helfand et al, compensates for the loss of the Ser130 hydroxyl group by donating a proton to the ␤-lactam nitrogen (153,157,180,408,438).…”

“…The decreased catalytic efficiencies seen in the TEM and SHV variants are likely a result of perturbations caused by the repositioned water molecule. These perturbations vary somewhat between SHV and TEM Ser130Gly (e.g., reorientation of Ser70 and Lys73 in SHV and TEM Ser130Gly, respectively), but both involve changes in hydrogen bonding networks in the active site, which are modified to accommodate the new water molecule (397,408).…”