Beta-lactamase (Class B1)

 

Beta-lactamase is a key enzyme in antibiotic resistance, catalysing the cleavage of the essential beta-lactam ring structure in penicillin and cephalosporinase type antibiotics. Substrate specificity varies considerably within the beta-lactamases, with some enzymes preferring penicillins and some cephalosporins.

Alongside the class B beta-lactamases, described here, there also exist three other classes of beta-lactam cleaving enzymes (A,C and D). These groups of enzymes use a serine nucleophilic mechanism, while class B uses a mono/dimetallic zinc mechanism. This entry describes the dimetallic mechanism. There is considerable variety in substrate specificity between the classes, although class C enzymes tend to have a high cephalosporinase activity

 

Reference Protein and Structure

Sequence
P25910 UniProt (3.5.2.6) IPR001279 (Sequence Homologues) (PDB Homologues)
Biological species
Bacteroides fragilis (Bacteria) Uniprot
PDB
1znb - METALLO-BETA-LACTAMASE (1.85 Å) PDBe PDBsum 1znb
Catalytic CATH Domains
3.60.15.10 CATHdb (see all for 1znb)
Cofactors
Zinc(2+) (2), Water (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:3.5.2.6)

beta-lactam
CHEBI:35627ChEBI
+
water
CHEBI:15377ChEBI
→
substituted beta-amino acids
CHEBI:33705ChEBI
Alternative enzyme names: Beta-lactamase A, B, C, Beta-lactamase AME I, Beta-lactamase I-III, Ampicillinase, Cephalosporin-beta-lactamase, Cephalosporinase, Exopenicillinase, Neutrapen, Penicillin beta-lactamase, Penicillin amido-beta-lactamhydrolase, Penicillinase, Penicillinase I, II,

Enzyme Mechanism

Introduction

A mechanism has been proposed by Fabiane et al (PMID:9730812). The beta-lactam carbonyl interacts with zinc1 polarising the bond and enhancing its susceptibility to nucleophilic attack. A zinc1 associated water/hydroxide is the nucleophile which attacks the beta-lactam carbonyl carbon. The substrate carboxylate moiety interacts with zinc2 and lysine 184. Asparagine 193 and zinc1 stabilise the oxyanion. An incoming water molecule is activated by the zinc ions and deprontonated by the intermediate.

Catalytic Residues Roles

UniProt PDB* (1znb)
Asn193 Asn193(176)A Stabilises the anionic tetrahedral intermediate. hydrogen bond donor, electrostatic stabiliser
Asp103, Cys181, His99, His101, His223, His162 Asp103(86)A, Cys181(164)A, His99(82)A, His101(84)A, His223(206)A, His162(145)A Binds one of the Zn(II) ions. metal ligand
Lys184 Lys184(167)A Binds the carboxylate group of the substrate, helping to stabilise the intermediate and correctly position the substrate. steric role, electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

bimolecular nucleophilic substitution, overall reactant used, decyclisation, intermediate formation, proton transfer, overall product formed, intermediate terminated, rate-determining step

References

  1. Fabiane SM et al. (1998), Biochemistry, 37, 12404-12411. Crystal Structure of the Zinc-Dependent β-Lactamase fromBacillus cereusat 1.9 Å Resolution:  Binuclear Active Site with Features of a Mononuclear Enzyme†,‡. DOI:10.1021/bi980506i. PMID:9730812.
  2. Spencer J et al. (2005), J Am Chem Soc, 127, 14439-14444. Antibiotic Recognition by Binuclear Metallo-β-Lactamases Revealed by X-ray Crystallography#. DOI:10.1021/ja0536062. PMID:16218639.
  3. Wang Z et al. (1999), Curr Opin Chem Biol, 3, 614-622. Metallo-β-lactamase: structure and mechanism. DOI:10.1016/s1367-5931(99)00017-4. PMID:10508665.
  4. Li Z et al. (1999), Protein Sci, 8, 249-252. For the record: Structural consequences of the active site substitution Cys181 → Ser in metallo-β-lactamase from bacteroides fragilis. DOI:10.1110/ps.8.1.249. PMID:10210203.
  5. Wang Z et al. (1999), Biochemistry, 38, 10013-10023. On the Mechanism of the Metallo-β-lactamase fromBacteroides fragilis†. DOI:10.1021/bi990356r. PMID:10433708.
  6. Fitzgerald PM et al. (1998), Biochemistry, 37, 6791-6800. Unanticipated Inhibition of the Metallo-β-lactamase fromBacteroidesfragilisby 4-Morpholineethanesulfonic Acid (MES):  A Crystallographic Study at 1.85-Å Resolution‡. DOI:10.1021/bi9730339. PMID:9578564.

Step 1. Zinc activated water initiates a nucleophilic attack on the carbonyl of the beta-lactam ring, breaking the C-N bond in a substitution reaction.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn193(176)A hydrogen bond donor, electrostatic stabiliser
Cys181(164)A metal ligand
His223(206)A metal ligand
Asp103(86)A metal ligand
His99(82)A metal ligand
His162(145)A metal ligand
His101(84)A metal ligand
Lys184(167)A steric role, electrostatic stabiliser

Chemical Components

ingold: bimolecular nucleophilic substitution, overall reactant used, decyclisation, intermediate formation

Step 2. The negatively charged nitrogen group then deprotonates an incoming, zinc-activated water molecule.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn193(176)A hydrogen bond donor
Cys181(164)A metal ligand
His223(206)A metal ligand
Asp103(86)A metal ligand
His162(145)A metal ligand
His101(84)A metal ligand
His99(82)A metal ligand
Lys184(167)A steric role, electrostatic stabiliser

Chemical Components

proton transfer, overall reactant used, overall product formed, intermediate terminated, rate-determining step
Download: Image, Marvin File

Step 1. Zinc activated water initiates a nucleophilic attack on the carbonyl of the beta-lactam ring, breaking the C-N bond in a substitution reaction.

Step 2. The negatively charged nitrogen group then deprotonates an incoming, zinc-activated water molecule.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Gail J. Bartlett, Sophie T. Williams, Craig Porter, Katherine Ferris