BETA-LACTAMASES

β-lactamases are the enzymes that brings about the hydrolysis of beta-lactam ring present in related group of antibiotics causing microbial resistance which was previously susceptible to the very group of antibiotics, eg. Penicillin. First bacterial enzyme reported to destroy Penicillin belonged to the β-lactamase of Escherichia coli in 1940 [1]. The same β-lactamase is now called the AmpC β-lactamase. These β-lactamases destroys the ability of β-lactam antibiotics.

The various classes of  β-lactam antibiotics include the following:

a) Penicillins: penicillin G, penicillin V, methicillin ,oxacillin, nafcillin, ampicillin, amoxicillin and carbenicillin

b) Cephalosporin: cephalothin; cefamandole, cefotaxime

c) Carbapenems: primaxin

d) Monobactams: aztreonam

Mechanism of action of Beta lactam antibiotics

The β-lactam antibiotics inhibits cell wall synthesis.These are characterized by its four-membered, nitrogen-containing β-lactam ring at the core of their structures. Beta lactam antibiotics target a group of enzymes found anchored in the cell membrane called as  penicillin-binding proteins or PBPs. PBPs are involved in the cross-linking of the bacterial cell wall. The beta-lactam ring portion of this group of antibiotics binds to these different PBPs, rendering them unable to perform their role in cell wall synthesis.  This then leads to death of the bacterial cell due to osmotic instability or autolysis. 

β-lactamases

β-Lactamases can be broadly divided into enzymes with a serine residue at the active site and metalloenzymes with zinc ion as a cofactor [2,3].

β-Lactamases are most commonly classified according to two general schemes: the Ambler molecular classification scheme and the Bush-Jacoby-Medeiros functional classification system [4]. 

The Ambler scheme (1980) divides β-lactamases into four major classes (A to D). This classification scheme is based upon protein homology (amino acid similarity) rather than  phenotypic characteristics. In the Ambler classification scheme, β-lactamases of classes A, C, and D are serine β-lactamases. In contrast, the class B enzymes are metallo-β-lactamases[5]. Thus, the Ambler scheme is a molecular classification.

The Bush-Jacoby-Medeiros classification scheme groups β-lactamases according to functional similarities (substrate and inhibitor profile) consisting of four main groups and multiple subgroups. This classification scheme is of much clinically relevant as it considers β-lactamase inhibitors and β-lactam substrates [5]. Thus, Bush-Jacoby-Medeiros classification is  a functional classification.

Mechanism by which β-lactamases resist β-lactam antibiotics

Broadly there are two β-lactamases (having serine residues or metalloenzyme with zinc in their active site) which hydrolyze and develop β-lactam antibiotics resistance. They can be described briefly as follow:

i) β-lactamases with serine residues in their active site

The  β-lactam ring is attracted to the active site of the enzyme forming hydrogen bond between negatively charged carboxylate or similar charged group with β-lactamases. Then, the antibiotics is acylated with enzyme forming β-lactam-β-lactamase complex and then a water molecule is activated which again revert the complex in its deacylated state. In this process, the configuration of  β-lactam ring is changed from closed to opened state and thus the antibiotics now become inactive while the enzyme becomes free and active.

i) β-lactamases with metalloenzyme residues with zinc in their active site

Metallo-β-lactamases require at least one or usually two zinc ions. The ions forms a bridge with the help of hydroxide ion which further facilitates nucleophilic attack on the carbonyl oxygen atom present in β-lactam ring. This leads to the hydrolysis and inactivation of the antibiotic as the configuration of β-lactam ring is changed from closed to opened state as mentioned earlier.

Usually there are two zinc ions. However, one zinc ion is held in place by three histidine residues. On contrary, many MBLs contain a second zinc binding site. There will be ligand formation between zinc and aspartate, cysteine and/or histidine. Moreover, histidine also assist in hydrolysis process [11,12].

Thus, β-lactamases contain either a reactive serine residues or at least one catalytically functional divalent zinc atom. 

There are now reports of emerging new types of β-lactamases including extended spectrum beta-lactamases (ESBLs), AmpC β-lactamases, and metallo β-lactamases [6-8]. These enzymes are able to hydrolyze broadspectrum cephalosporins including ceftazidime, ceftriaxone, cefepime, and monobactams (aztreonam and cephamycin). AmpC β-lactamases are resistant to 7-alfa methoxy cephalosporin and monobactams.

A comparison between the classification schemes of β-lactamases are illustrated below [8,9]: 

S. No.	Catalytic Site	Ambler Class	Bush-Jacoby Medeiros group	Type of β-lactamase	Examples
1	Serine	A	2a	Penicillinases	Penicillinases produced by Gram-positive bacteria
			2b	Penicillinases	TEM-1, TEM-2 and SHV-1
			2be	ESBLs	TEM-10, CTX-M, GES-1 and SHV-2
			2br	Inhibitor-resistant	TEM-30 and SHV-72
			2c	Penicillinases	PSE (CARB)
			2f	Carbapenemases	KPC, SME, NMC-A and GES-2
2	Metallo	B	3	Carbapenemases	IMP, VIM and NDM
3	Serine	C	1	Cephalosporinases	Chromosomal AmpC, CMY, ACT-1 and DHA
4	Serine	D	2d	Oxacillinases	OXA-1/30, OXA-10, OXA-23 and OXA-48

REFERENCES

1. Abraham, E. P., and E. Chain. 1940. An enzyme from bacteria able to destroy penicillin. Nature 146:837.

2. Joris B, Ghuysen JM, Dive G, et al. The active-site-serine penicillin-recognizing enzymes as members of the Streptomyces R61 DD-peptidase family. Biochem J 1988;250:313-24. 

3. Garau G, García-Sáez I, Bebrone C, et al. Update of the standard numbering scheme for class B b-lactamases. Antimicrob Agents 

Chemother 2004;48:2347-9.

4. Ambler, R. P., A. F. Coulson, J. M. Frere, J. M. Ghuysen, B. Joris, M. Forsman, R. C. Levesque, G. Tiraby, and S. G. Waley. 1991. A standard numbering scheme for the class A beta-lactamases. Biochem. J. 276:269–270.

5. Paterson D.L. and Bonomo R.A. 2005. Extended-Spectrum β-Lactamases: a Clinical Update. Clinical Microbiology Reviews. ASM. Vol. 18, No. 4, p. 657–686.

6.Babic M, Hujer AM, Bonomo RA. What’s new in antibiotic resistance? Focus on beta-lactamases. Drug Resist Updat 2006 Jun;9(3):142e56.

7.Bredford P. Extended-spectrum ß-lactamases in the 21st century:characterization, epidemiology and detection of this important resistance threat. Clin Microbiol Rev; 2001:933e51.

8. Paterson DL. Resistance in gram-negative bacteria: enterobacteriaceae.
Am J Med 2006 Jun;119(6):S20e8.

9. Bush, K. Past and present perspectives on β-lactamases. Antimicrob. Agents Chemother. 62, e01076–18 (2018).

10. Reading, C. & Cole, M. Clavulanic acid: a beta-lactamase-inhiting beta-lactam from Streptomyces clavuligerus. Antimicrob. Agents Chemother. 11, 852–857 (1977).

11. Palzkill, T. Metallo-β-lactamase structure and function. Ann. NY Acad. Sci 1277, 91–104 (2013).

12. Bush, K., Bradford, P.A. Interplay between β-lactamases and new β-lactamase inhibitors. Nat Rev Microbiol 17, 295–306 (2019). https://doi.org/10.1038/s41579-019-0159-8.