β-Lactam antibiotics are the most frequently prescribed antibiotics, including penicillins, cephalosporins, and carbapenems [1,2]. Their structures share the four-membered cyclic amide called the β-lactam ring. The carbonyl carbon in the ring is covalently linked to the nucleophilic serine of penicillin-binding proteins (PBPs), catalysing the peptidoglycan cross-linking process to inhibit bacterial cell wall formation. The production of β-lactamases is the most prevailing drug-resistant mechanism of bacterial pathogens since the enzymes inactivate β-lactam antibiotics by breaking the amide bond in the β-lactam ring. β-Lactamases can be grouped into four classes (A, B, C, and D) based on their sequence similarity. Class A, C, and D β-lactamases have a serine nucleophile in their active sites, while class B β-lactamases require zinc ions to activate a nucleophilic water molecule for the cleavage of the β-lactam ring [3,4].

Penicillins and early-generation cephalosporins are easily inactivated by β-lactamases. To escape inactivation by β-lactamases, oxyimino cephalosporins including cefotaxime and ceftazidime, have been developed. They have bulky oxyimino groups, such as a 2-(2-aminothiazole-4-yl)-2-oxyimino substituent at position C7 of the β-lactam nucleus [5]. Initially, oxyimino cephalosporins were poor substrates for β-lactamases, but their clinical usage gave rise to extended-spectrum β-lactamases (ESBL) that can hydrolyse oxyimino β-lactams [6]. Such patterns have also been identified among class A β-lactamases. Class A β-lactamases are the most common type of β-lactamases found in clinical isolates, and they can be classified into three subgroups according to substrate specificity: penicillinases, cephalosporinases, and carbapenemases [7,8]. Natural mutants of the well-known penicillinases TEM-1, TEM-2, and SHV-1, harbouring point mutations (E104K, R164S, M182T, A237T, G238S, and E240K), can hydrolyse oxyimino cephalosporins [9,10]. In addition, novel groups of class A ESBLs that exhibit low sequence identity to TEM and SHV-type ESBLs have been reported worldwide. Among them, CTX-M-type β-lactamases hydrolyse cefotaxime and ceftriaxone [11], and other clinically relevant ESBL enzymes (e.g., KPC, SME, IMI/NMC-A, and certain variants of GES) hydrolyse carbapenems as well as penicillins and cephalosporins [12]. These class A carbapenemases are a great threat to public health in that they hydrolyse carbapenems, “antibiotics of last resort” to treat multidrug-resistant bacterial infections, [13,14] and they are often encoded on plasmids transmissible to other bacterial species [15]. Consequently, the substrate profile of class A enzymes covers all the clinically important cephalosporins and carbapenems, indicating that class A enzymes are directly related to the current antibiotic resistance crisis [16].

Previous structural and kinetic studies on TEMs, SHVs, CTX-Ms, KPCs, etc., revealed the shared catalytic mechanism among class A β-lactamases [4,10,17]. The nucleophilic Ser70 assisted by either Lys73 or Glu166 serving as a general base attacks the carbonyl carbon of the β-lactam ring, leading to the formation of the acyl-enzyme intermediate. Afterwards, Glu166 activates a water molecule for deacylation of the covalent intermediate. In addition to the key residues, other conserved residues contribute to catalysis. Ser130 mediates proton shuttling between Lys73 and the leaving group nitrogen during acylation. Asp170 is important in the positioning of the deacylating water. The backbone -NH groups of residues at positions 70 and 237 form the oxyanion hole that stabilizes a negatively charged transition-state oxyanion in the hydrolytic reaction. In addition, Asn132, Lys234, and Ser/Thr235 have been implicated in substrate binding and transition state stabilization. Despite the shared catalytic machinery, however, it was also highlighted that subtle sequence variations in the active site alter its size, shape, and flexibility [10], conferring differences in their substrate spectra [4]. Therefore, it is important to investigate local structural differences at the atomic level to reveal the molecular mechanism for the divergence of class A β-lactamases.

Stenotrophomonas spp. are Gram-negative bacteria that are found throughout the environment, particularly in close association with plants and soil [18]. Rich microbial communities and the spread of bacterial pathogens in their biotopes subject them to acquire multidrug-resistant profiles [19]. For example, the most dominant species, Stenotrophomonas maltophilia, has been reported to be a nosocomial multidrug-resistant pathogen leading to high mortality in immunocompromised patients [20]. S. maltophilia is resistant to all types of β-lactam antibiotics, which arises primarily from the production of two ESBL β-lactamases, a Zn2+-dependent class B enzyme (L1) and a class A β-lactamase (L2) [21]. Here, we present the biochemical and structural characterization of an uncharacterized class A β-lactamase from Stenotrophomonas sp. KCTC 12332 that shows 54.31% sequence identity to the L2 class A enzyme. This enzyme prefers cefaclor as a substrate (see below), and thus, it is named CESS-1 (for cefaclor preferring extended-spectrum β-lactamase from Stenotrophomonas sp.). Our study, based on the structures of CESS-1 acylated by three distinct substrates, provides mechanistic insights into the substrate discrimination of CESS-1. Additionally, we aim to extend our knowledge into an uncharacterized class A enzyme to elucidate the molecular mechanism for the divergence of class A β-lactamases.