Class D β-Lactamases: A Reappraisal after Five Decades

Abstract

Conspectus Despite 70 years of clinical use, β-lactam antibiotics still remain at the forefront of antimicrobial chemotherapy. The major challenge to these life-saving therapeutics is the presence of bacterial enzymes (i.e., β-lactamases) that can hydrolyze the β-lactam bond and inactivate the antibiotic. These enzymes can be grouped into 4 classes (A-D). Among the most genetically diverse are the class D β-lactamases. In this class are β-lactamases that can inactivate the entire spectrum of β- lactam antibiotics (penicillins, cephalosporins, and carbapenems). Class D β-lactamases are mostly found in Gram-negative bacteria such as Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, and Acinetobacter baumannii. The active-sites of class D β-lactamases contain an unusual N-carboxylated lysine post-translational modification. A strongly hydrophobic active-site helps create the conditions that allow the lysine to combine with CO2, and the resulting carbamate is stabilized by a number of hydrogen bonds. The carboxy-lysine plays a symmetric role in the reaction, serving as a general base to activate the serine nucleophile in the acylation reaction, and the deacylating water in the second step. There are more than 250 class D β-lactamases described, and the full set of variants shows remarkable diversity with regard to substrate binding and turnover. Narrow-spectrum variants are most effective against the earliest generation penicillins and cephalosporins such as ampicillin and cephalothin. Extended-spectrum variants (also known as extended-spectrum β-lactamases, ESBLs) pose a more dangerous clinical threat as they possess a small number of substitutions that allow them to bind and hydrolyze later generation cephalosporins that contain bulkier side-chain constituents (eg. cefotaxime, ceftazidime, and cefepime). Mutations that permit this versatility seem to cluster in the area surrounding an active-site tryptophan resulting in a widened active-site to accommodate the oxyimino side-chains of these cephalosporins. More concerning are the class D β-lactamases that hydrolyze clinically-important carbapenem β-lactam drugs (e.g., imipenem). Whereas carbapenems irreversibly acylate and inhibit narrow- spectrum β-lactamases, class D carbapenemases are able to recruit and activate a deacylating water. The rotational orientation of the C6 hydroxyethyl group found on all carbapenem antibiotics likely plays a role in whether the deacylating water is effective or not. Inhibition of class D β-lactamases is a current challenge. Commercially available inhibitors that are active against other classes of β-lactamases are ineffective against class D enzymes. On the horizon are several compounds, consisting of both β-lactam derivatives and non-β-lactams, that have the potential of providing novel leads to design new mechanism-based inactivators that are effective against the class D enzymes. Several act synergistically when given in combination with a β-lactam antibiotic, and others show a unique mechanism of inhibition that is d...

Clinical Variants of the Native Class D β-Lactamase of Acinetobacter baumannii Pose an Emerging Threat through Increased Hydrolytic Activity against Carbapenems

“…A wide variety of ␤-lactamases from all four classes (A to D) have been shown to be present in A. baumannii (8), but class D carbapenemases have received the most attention in recent years (9). Most class D carbapenemases are characterized by very high affinity for carbapenem substrates, coupled to relatively weak catalytic turnover rates (10). Five subfamilies of class D carbapenemases have been identified in A. baumannii, namely, OXA-23, OXA-24/40, OXA-51, OXA-58, and OXA-143 (1).…”

“…For most of these enzymes, structures have been determined with substrates or inhibitors bound in the active site (21)(22)(23)(24)(25)(26). Like class A and class C ␤-lactamases, members of class D make use of a serine-nucleophile acylation/deacylation double-displacement mechanism to hydrolyze the ␤-lactam ring (10). Two features are unique to class D enzymes, however.…”

Structural Origins of Oxacillinase Specificity in Class D β-Lactamases

“…Alternate conformations of Ser115 and, in some cases, even more significant movement of this residue have been noted in structures of other OXA enzymes (24,(28)(29)(30)(31). Ser115 is implicated in playing a role in proton transfer in the hydrolytic mechanism (28,32,33). In acylation, Ser115 is likely involved in a relay system that transfers a proton from the general base (i.e., the carboxy-Lys70) to the leaving group nitrogen of the lactam ring, and in deacylation Ser115 could transfer this proton to Ser67 (33).…”

“…Ser115 is implicated in playing a role in proton transfer in the hydrolytic mechanism (28,32,33). In acylation, Ser115 is likely involved in a relay system that transfers a proton from the general base (i.e., the carboxy-Lys70) to the leaving group nitrogen of the lactam ring, and in deacylation Ser115 could transfer this proton to Ser67 (33). Ser115 must rotate to accomplish this.…”

The Different Inhibition Mechanisms of OXA-1 and OXA-24 β-Lactamases Are Determined by the Stability of Active Site Carboxylated Lysine

“…The formation of the carboxylated lysine is reversible (8). Low pH or mutation of hydrophobic residues surrounding the carboxylated lysine, such as Val-117 (OXA-1) 3 or Trp-154 (OXA-10), results in decarboxylation of that lysine and loss of enzyme activity, notably deacylation (9 -12), whereas the addition of bicarbonate can reactivate the enzyme by recarboxylation of the lysine (8,12,13).…”