Hydrolysis of Third-generation Cephalosporins by Class C β-Lactamases

Abstract

Bacterial resistance to the third-generation cephalosporins is an issue of great concern in current antibiotic therapeutics. An important source of this resistance is from production of extended-spectrum (ES) ␤-lactamases by bacteria. The Enterobacter cloacae GC1 enzyme is an example of a class C ES ␤-lactamase. Unlike wild-type (WT) forms, such as the E. cloacae P99 and Citrobacter freundii enzymes, the ES GC1 ␤-lactamase is able to rapidly hydrolyze third-generation cephalosporins such as cefotaxime and ceftazidime. To understand the basis for this ES activity, m-nitrophenyl 2-(2-aminothiazol-4-yl)-2-[(Z)-methoxyimino]acetylaminomethyl phosphonate has been synthesized and characterized. This phosphonate was designed to generate a transition state analog for turnover of cefotaxime. The crystal structures of complexes of the phosphonate with both ES GC1 and WT C. freundii GN346 ␤-lactamases have been determined to high resolution (1.4 -1.5 Å). The serine-bound analog of the tetrahedral transition state for deacylation exhibits a very different binding geometry in each enzyme. In the WT ␤-lactamase the cefotaxime-like side chain is crowded against the ⍀ loop and must protrude from the binding site with its methyloxime branch exposed. In the ES enzyme, a mutated ⍀ loop adopts an alternate conformation allowing the side chain to be much more buried. During the binding and turnover of the cefotaxime substrate by this ES enzyme, it is proposed that ligand-protein contacts and intra-ligand contacts are considerably relieved relative to WT, facilitating positioning and activation of the hydrolytic water molecule. The ES ␤-lactamase is thus able to efficiently inactivate third-generation cephalosporins.␤-Lactam antibiotics are widely used because of their effectiveness and safety. However, ␤-lactam-resistant bacteria have become widespread. The main cause of this resistance is the production of ␤-lactamases, which hydrolyze the amide bond in the ␤-lactam ring. ␤-Lactamases are grouped into four classes, A-D, on the basis of amino acid sequence. Although class B ␤-lactamases are metallo-␤-lactamases, class A, C, and D ␤-lactamases are serine-reactive hydrolases that function by acylation and deacylation steps employing tetrahedral intermediates (1, 2) (see Reaction 1).To overcome ␤-lactam resistance, third-generation cephalosporins such as cefotaxime (1), ceftazidime (2), and ceftriaxon have been employed since the 1980s because of their relative stability to serine ␤-lactamases and their broad antibacterial spectrum. Third-generation cephalosporins typically contain a bulky group such as a 2-(2-aminothiazole-4-yl)-2-oxyimino substituent (R 1 ) at position C7 of the cephalosporin nucleus.From the kinetic perspective, third-generation cephalosporins show high K m and low k cat values for class A and D ␤-lactamases, and low K m (K i ) and low k cat values for class C enzymes. This behavior suggests that these cephalosporins cannot effectively bind to the active site of wild-type (WT) 1 class A ␤-lactamases and acylate them...

Crystal Structure of Mox-1, a Unique Plasmid-Mediated Class C β-Lactamase with Hydrolytic Activity towards Moxalactam

“…Among the enzymes whose kinetic properties have been studied, the AmpC ␤-lactamase from Enterobacter cloacae P99 is the most similar to Mox-1 in amino acid sequence with 45% identity, and the RMSD is 3.03 Å. The RMSD value between Mox-1 and an extended-spectrum enzyme (GC1) from E. cloacae (45% identical in primary sequence) was 3.29 Å. GC1 is 98% identical to P99, and it was reported that GC1 acquired its extended substrate specificity by the insertion of 3 amino acids in the ⍀ loop (22). The large RMSD suggests that the extended-spectrum ␤-lactamase activity is different between Mox-1 and GC1.…”

Avibactam and Class C β-Lactamases: Mechanism of Inhibition, Conservation of the Binding Pocket, and Implications for Resistance

“…Given the conservation of the avibactam binding pocket residues and the importance of these residues in ␤-lactam recognition and catalysis (25)(26)(27)(28)(29), we investigated whether any variations in these might compromise the inhibition potency of avibactam while still allowing hydrolysis of the ␤-lactam drug and thus result in resistance. Spontaneous resistance frequency experiments were carried out in several isolates carrying class C ␤-lactamases, with an initial focus on Enterobacteriaceae spp.…”

Substrate Deacylation Mechanisms of Serine-.BETA.-lactamases

“…However, as seen in X-ray crystallographic analysis structures of class C b-lactamase, [48][49][50][51][52][53] there is no acidic amino acid residue corresponding to Glu166 in class A in the active site, while a residue corresponding to Lys73 in class A is seen (Lys67). In place of Glu166 in class A, a tyrosine residue (Tyr150) exists in the active site of class C. Some studies 8,48,54) have suggested that the side chain of Tyr150 is in a deprotonated state due to the special surrounding environment of class C and plays a role as a general base.…”