β-Lactamases, the major resistance determinant for β-lactam antibiotics in Gram-negative bacteria, are ancient enzymes whose origins can be traced back millions of years ago. These well-studied enzymes, currently numbering almost 2,800 unique proteins, initially emerged from environmental sources, most likely to protect a producing bacterium from attack by naturally occurring β-lactams. Their ancestors were presumably penicillin-binding proteins that share sequence homology with β-lactamases possessing an active-site serine. Metallo-β-lactamases also exist, with one or two catalytically functional zinc ions. Although penicillinases in Gram-positive bacteria were reported shortly after penicillin was introduced clinically, transmissible β-lactamases that could hydrolyze recently approved cephalosporins, monobactams, and carbapenems later became important in Gram-negative pathogens. Nomenclature is based on one of two major systems. Originally, functional classifications were used, based on substrate and inhibitor profiles. A later scheme classifies β-lactamases according to amino acid sequences, resulting in class A, B, C, and D enzymes. A more recent nomenclature combines the molecular and biochemical classifications into 17 functional groups that describe most β-lactamases. Some of the most problematic enzymes in the clinical community include extended-spectrum β-lactamases (ESBLs) and the serine and metallo-carbapenemases, all of which are at least partially addressed with new β-lactamase inhibitor combinations. New enzyme variants continue to be described, partly because of the ease of obtaining sequence data from whole-genome sequencing studies. Often, these new enzymes are devoid of any phenotypic descriptions, making it more difficult for clinicians and antibiotic researchers to address new challenges that may be posed by unusual β-lactamases.

Past and Present Perspectives on β-Lactamases.

The efficiency of beta-lactam antibiotics, which are among our most useful chemotherapeutic weapons, is continuously challenged by the emergence of resistant bacterial strains. This is most often due to the production of beta-lactamases by the resistant cells. These enzymes inactivate the antibiotics by hydrolysing the beta-lactam amide bond. The elucidation of the structures of some beta-lactamases by X-ray crystallography has provided precious insights into their catalytic mechanisms and revealed unsuspected similarities with the DD-transpeptidases, the bacterial enzymes which constitute the lethal targets of beta-lactams. Despite numerous kinetic, structural and site-directed mutagenesis studies, we have not completely succeeded in explaining the diversity of the specificity profiles of beta-lactamases and their surprising catalytic power. The solutions to these problems represent the cornerstones on which better antibiotics can be designed, hopefully on a rational basis.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2958.1995.tb02404.x

Beta‐lactamases and bacterial resistance to antibiotics

(1984). Penicillin-Sensitive Enzymes in Peptidoglycan Biosynthesis. CRC Critical Reviews in Microbiology: Vol. 11, No. 4, pp. 299-396.

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Penicillin-sensitive enzymes in peptidoglycan biosynthesis.

Structural data are now available for comparing a penicillin target enzyme, the D-alanyl-D-alanine-peptidase from Streptomyces R61, with a penicillin-hydrolyzing enzyme, the beta-lactamase from Bacillus licheniformis 749/C. Although the two enzymes have distinct catalytic properties and lack relatedness in their overall amino acid sequences except near the active-site serine, the significant similarity found by x-ray crystallography in the spatial arrangement of the elements of secondary structure provides strong support for earlier hypotheses that beta-lactamases arose from penicillin-sensitive D-alanyl-D-alanine-peptidases involved in bacterial wall peptidoglycan metabolism.

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On the origin of bacterial resistance to penicillin: comparison of a beta-lactamase and a penicillin target

The bacterial acyltransferases of the SxxK superfamily vary enormously in sequence and function, with conservation of particular amino acid groups and all-α and α/β folds. They occur as independent entities (free-standing polypeptides) and as modules linked to other polypeptides (protein fusions). They can be classified into three groups. The group I SxxK d,d-acyltransferases are ubiquitous in the bacterial world. They invariably bear the motifs SxxK, SxN(D), and KT(S)G. Anchored in the plasma membrane with the bulk of the polypeptide chain exposed on the outer face of it, they are implicated in the synthesis of wall peptidoglycans of the most frequently encountered (4→3) type. They are inactivated by penicillin and other β-lactam antibiotics acting as suicide carbonyl donors in the form of penicillin-binding proteins (PBPs). They are components of a morphogenetic apparatus which, as a whole, controls multiple parameters such as shape and size and allows the bacterial cells to enlarge and duplicate their particular pattern. Class A PBP fusions comprise a glycosyltransferase module fused to an SxxK acyltransferase of class A. Class B PBP fusions comprise a linker, i.e., protein recognition, module fused to an SxxK acyltransferase of class B. They ensure the remodeling of the (4→3) peptidoglycans in a cell cycle-dependent manner. The free-standing PBPs hydrolyze d,d peptide bonds. The group II SxxK acyltransferases frequently have a partially modified bar code, but the SxxK motif is invariant. They react with penicillin in various ways and illustrate the great plasticity of the catalytic centers. The secreted free-standing PBPs, the serine β-lactamases, and the penicillin sensors of several penicillin sensory transducers help the d,d-acyltransferases of group I escape penicillin action. The group III SxxK acyltransferases are indistinguishable from the PBP fusion proteins of group I in motifs and membrane topology, but they resist penicillin. They are referred to as Penr protein fusions. Plausible hypotheses are put forward on the roles that the Penr protein fusions, acting as l,d-acyltransferases, may play in the (3→3) peptidoglycan-synthesizing molecular machines. Shifting the wall peptidoglycan from the (4→3) type to the (3→3) type could help Mycobacterium tuberculosis and Mycobacterium leprae survive by making them penicillin resistant.

Biochemistry and comparative genomics of SxxK superfamily acyltransferases offer a clue to the mycobacterial paradox: presence of penicillin-susceptible target proteins versus lack of efficiency of penicillin as therapeutic agent.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2876%2980770-3

Joël Vandekerkhove

Papers

Occurrence of a serine residue in the penicillin-binding site of the exocellular DD-carboxy-peptidase-transpeptidase from Streptomyces R61.