Crystal structure of a preacylation complex of the β-lactamase inhibitor sulbactam bound to a sulfenamide bond-containing thiol-β-lactamase

Abstract

The rise of inhibitor-resistant and other β-lactamase variants is generating an interest in developing new β-lactamase inhibitors to complement currently available antibiotics. To gain insight into the chemistry of inhibitor recognition, we determined the crystal structure of the inhibitor preacylation complex of sulbactam, a clinical β-lactamase inhibitor, bound in the active site of the S70C variant of SHV-1 β-lactamase, a resistance enzyme that is normally present in Klebsiella pneumoniae. The S70C mutation was designed to affect the reactivity of that catalytic residue to allow for capture of the preacylation complex. Unexpectedly, the 1.45 Å resolution inhibitor complex structure revealed that residue C70 is involved in a sulfenamide bond with K73. Such a covalent bond is not present in the wild-type SHV-1 or in an apo S70C structure also determined in this study. This bond likely contributed significantly to obtaining the preacylation complex with sulbactam due to further decreased reactivity toward substrates. The intact sulbactam is positioned in the active site such that its carboxyl moiety interacts with R244, S130, and T235 and its carbonyl moiety is situated in the oxyanion hole. To our knowledge, in addition to being the first preacylation inhibitor β-lactamase complex, this is also the first observation of a sulfenamide bond between a cysteine and lysine in an active site. Not only could our results aid, therefore, structure-based inhibitor design efforts in class A β-lactamases, but the sulfenamide-bond forming approach to yield preacylation complexes could also be applied to other classes of β-lactamases and penicillin-binding proteins with the SXXK motif.

Figure 1

Chemical structures of three clinically available inhibitors: (A) sulbactam, (B) clavulanic acid, (C) tazobactam and two substrates: (D) cephalothin, (E) cefamandole.

Figure 2

Intact, unreacted sulbactam in the S70C active site. (A) F_o − F_c density contoured at 2.75σ; (B) 2F_o − F_c density contoured at 1.0σ.

Figure 3

Stereo figure depicting sulbactam in the active site of S70C.

Figure 4

Superposition of S70C:sulbactam (gray), apo S70C (green) and apo wtSHV-1 (cyan). Shown are residues within hydrogen bonding distance of the carboxylic acid group of sulbactam, multiple conformations for S130 and K234 and similarity of R244 and T235 conformations across the three structures. Also depicted are oxyanion hole occupants: partially occupied water molecules in the apo wtSHV-1 (cyan sphere) and S70C:sulbactam (gray sphere, mostly eclipsed by cyan sphere) structures and lack of water in apo S70C structure because C70 sulfur has shifted toward that position. Superpositions were performed using SSM Superpose utility in COOT.

Figure 5

Substrate preacylation complex superpositions show similarities and discrepancies in active site interactions: (A) S70C:sulbactam (gray) and AmpC:cephalothin (purple, 1KVL). Residues 69–73, 132, and 234–238 were aligned with residues 63–67, 152, and 315–219, respectively. (B) S70C:sulbactam (gray) and BlaC:cefamandole (pink, 3NY4). Residues 69–73, 132, and 234–238 were aligned with residues 83–87, 144, and 250–254, respectively. Superpositions were performed using the LSQKAB program in the CCP4 suite.