Exploring the inhibition of CTX-M-9 by beta-lactamase inhibitors and carbapenems

Abstract

Currently, CTX-M β-lactamases are among the most prevalent and most heterogeneous extended-spectrum β-lactamases (ESBLs). In general, CTX-M enzymes are susceptible to inhibition by β-lactamase inhibitors. However, it is unknown if the pathway to inhibition by β-lactamase inhibitors for CTX-M ESBLs is similar to TEM and SHV β-lactamases and why bacteria possessing only CTX-M ESBLs are so susceptible to carbapenems. Here, we have performed a kinetic analysis and timed electrospray ionization mass spectrometry (ESI-MS) studies to reveal the intermediates of inhibition of CTX-M-9, an ESBL representative of this family of enzymes. CTX-M-9 β-lactamase was inactivated by sulbactam, tazobactam, clavulanate, meropenem, doripenem, ertapenem, and a 6-methylidene penem, penem 1. K(i) values ranged from 1.6 ± 0.3 μM (mean ± standard error) for tazobactam to 0.02 ± 0.01 μM for penem 1. Before and after tryptic digestion of the CTX-M-9 β-lactamase apo-enzyme and CTX-M-9 inactivation by inhibitors (meropenem, clavulanate, sulbactam, tazobactam, and penem 1), ESI-MS and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identified different adducts attached to the peptide containing the active site Ser70 (+52, 70, 88, and 156 ± 3 atomic mass units). This study shows that a multistep inhibition pathway results from modification or fragmentation with clavulanate, sulbactam, and tazobactam, while a single acyl enzyme intermediate is detected when meropenem and penem 1 inactivate CTX-M-9 β-lactamase. More generally, we propose that Arg276 in CTX-M-9 plays an essential role in the recognition of the C(3) carboxylate of inhibitors and that the localization of this positive charge to a "region of the active site" rather than a specific residue represents an important evolutionary strategy used by β-lactamases.

Fig. 1.

Chemical structures of indicator substrate (nitrocefin [1]) and β-lactamase inhibitors (clavulanate [2], sulbactam [3], tazobactam [4], meropenem [5; with the R₂ side chain at the C₂ position], ertapenem [6], doripenem [7], and penem 1 [8]) used in this study. The C atom numbering system is shown for meropenem.

Fig. 2.

Deconvoluted mass spectra of CTX-M-9 β-lactamase (E, apo-enzyme) and CTX-M-9 β-lactamase with inhibitors (I) in a variable I/E ratio. Mass increases (products of inactivation) are shown. (A) CTX-M-9 β-lactamase alone; (B) CTX-M-9 with meropenem; (C) CTX-M-9 with penem 1; (D) CTX-M-9 β-lactamase with clavulanate; (E) CTX-M-9 β-lactamase with sulbactam; (F) CTX-M-9 with tazobactam. All measurements have an error of ±3 amu. For the commercially available inhibitors (clavulanate, sulbactam, and tazobactam), multiple products are shown.

Fig. 3.

Postulated chemical structures of products of inactivation shown in Fig. 2. (a and b) The two forms of the N terminus of CTX-M-9. Panel a shows the pyroglutamate form. (c and d) The two forms of meropenem as the acyl enzyme (panel d shows the meropenem minus the ethoxy group from the C₆ position). (e) The penem 1 acyl enzyme. In the case of the 52 ± 3 Da adduct shown in Fig. 2D, E, and F, we assigned the structure represented in panel i as the possible adduct for the structures shown in Fig. 2E and F (propynyl group). (f) It is also possible that the chemical moiety represented is present and represented in intermediates shown in Fig. 2D, E, and F. (g and h) The semialdlehye and aldehyde, respectively. (j) The 155-Da adduct.

Fig. 4.

UV difference spectroscopy data were obtained by subtraction of free inhibitor from CTX-M-9 with added inhibitor at t = 0. (a) UV difference spectroscopy of CTX-M-9 reacted with penem 1 (I/E of 100/1). (b) Meropenem (I/E of 1,000/1). Note the formation of a chromophore at 304 nm and 360 nm. In accordance with previous data for these inhibitors with the class A β-lactamase SHV, we tentatively assigned the chromophore at 304 nm to the hydrolysis of the β-lactam bond and the chromophore at 360 to the formation of the bicyclic aromatic ring system (2).

Fig. 5.

Molecular representations of CTX-M-9 and meropenem in the Δ² tautomeric form (a and b) and Δ¹ tautomeric form (c and d) docked in the active site of CTX-M-9. (a) The stable acyl enzyme species of meropenem and CTX-M-9 with the carbonyl oxygen of meropenem in the oxyanion hole formed by the amides of Ser70 and Ser237. In addition, the C₃ carboxylate makes hydrogen bonds with T235 through a water molecule and the delocalized positively charged guanidinium group of Arg276. In this acyl enzyme model, meropenem's substituent at the C₆ position interacts via hydrogen bonds to Asn132 and Asn104. (c) During the dynamic simulation, the carbonyl oxygen of meropenem flipped out of the oxyanion hole toward Lys73, making a hydrogen bond with its amine group. The carboxylate made a hydrogen bond network with Arg276 and Thr235 (the interaction mediated by a water molecule was replaced by a hydrogen bond interaction). The substituent at the C₆ position group showed increased flexibility during MDS, maintaining the interactions with Asn132 and/or Asn104. (b and d) Connolly surface diagrams of meropenem in CTX-M-9, colored by atom charge. These representations show a change in the shape and electropositivity of the enzyme active site in the region of Arg276 upon binding with the two forms of meropenem. The larger positively charged region found near Arg276 in panel b is replaced by a more localized region closer to the oxyanion hole in panel d, suggesting that Arg276 possesses a flexible side chain and that conformational changes occur with the enzyme upon binding meropenem.

Fig. 6.

Molecular representation of penem 1 prior to endo-trig cyclization and formation of the 7-membered 1,4-thiazepine ring having R stereochemistry at the new C₇ moiety, as seen with SHV-1 and penem 1, in the active site of CTX-M-9 (35). (a) The acyl enzyme with the carbonyl oxygen in the oxyanion hole formed by the amides of Ser70 and Ser237. Unlike the meropenem CTX-M-9 model presented in Fig. 5a, this model shows the C₃ carboxylate making a hydrogen bond with the -OH of Tyr105 and losing the interactions with Thr235 (∼5 Å) and Arg276 (∼6 Å). In addition, two water molecules are positioned such that they may bridge the interaction between the two residues and the C₃ carboxylate. (b) Connolly surface diagram of penem 1 in CTX-M-9.