P-Lactamase TEM

The class A P-lactamases are a subset of the active-site serine P-lactamases. TEM-1 P-lactamase is a class A enzyme encoded by the ft/ajEM-l gene that is present on the transposons Tn2 and Tn3 (Datta et al, 1965). Epidemiological studies have shown that TEM-1 is the most common plasmid-mediated P-lactamase and is therefore a major determinant of bacterial resistance to P-lactam antibiotics (Wiedemann et al, 1989). Compounding the problem of resistance is the discovery that TEM-1 mutant variants with altered substrate specificity have been identified in natural isolates (Jacoby and Medieros, 1991). These variant enzymes contain from one to three amino acid substitutions that enable the enzyme to hydrolyze the newer extended-spectrum cephalosporin antibiotics such as cefotaxime and ceftazidime (Jacoby and Medieros, 1991). Thus, the selective pressure of antibiotic therapy le s to die creation of new enzymes with expanded hydrolytic capabilities. 

Cephalosporins Extended spectrum P-lactamases chromosomal P-lactamases (AmpC) plasmid P-lactamases (TEM-1, SHV-1)... 

Class A Serine p-lactamases SHV-1 penicillinase in K. pneumoniae, and Koxy with activity against certain third generation cephalosporins in K. oxytoca BlaZ staphylococcal penicillinase TEM, SHV, VEB, PER and CTX-M penicillinases and ESBLs (P-lactamases with activity against third generation cephalosporins and aztreo-nam) KPC, IMI/NMC and SME carbapenemases... 

Figure 1 A stereoview image that depicts the active site of TEM-1 p-lactamase from coli. The protein is depicted in capped-sticks representation. Residues are shown in capped-sticks representation and coior-coded according to atom types (N, C, O, and H in blue, white, red and cyan, respectively). The red arrow points to Lys-73, and the white arrow points to Ciu-166.

Because of the significant role of TEM-1 P-lactamase and its mutant derivatives in antibiotic resistance, it is of interest to understand how the amino acid sequence of the enzyme establishes its structure and activity. We have determined the tolerance of each residue in TEM-1 P-lactamase to amino acid substitutions to identify those residues that make critical contributions to the structure and activity of the enzyme. The tolerance of each residue was determined by randomizing three to six contiguous codons to create a random library containing all possible amino acid substitutions for the region randomized (Palzicill and Botstein, 1992). Functional random mutants were then selected from the libraries and sequenced to identify permissible substitutions at each position. The sequences for each set of mutants allowed the importance of... 

To determine the identity of allowable substitutions at each residue position, the DNA sequence of an average of 9 functional random mutants from each library were determined. In total, 43 out of the 263 (16%) mutated residues are inferred to be critical for TEM-1 p-lactamase structure and function since only the wild type amino acid is found at these positions among the sequenced mutants. This set of essential residues includes catalytic residues and a number of other amino acids that are buried in the hydrophobic core of the enzyme. A detailed description and analysis of these results has been published elsewhere (Huang et al., 1996). 

A large number of class A P-lactamases have now been identified and sequenced and an alignment of 20 class A P-lactamases has been published (Ambler et al., 1991). These aligned sequences permit a comparison between the conserved amino acid residue positions among class A p-lactamases and the conserved positions among the functional random mutants in TEM P-lactamase (Fig. 2). In general, there is agreement between the tolerance of a residue in TEM P-lactamase to amino acid substitutions and the amount a position is substituted in... 

Figure 2. Comparison of sequence variability among functional TEM-1 p-lactamase mutants and twenty aligned class A P-lactamases. The wild type TEM-1 -lactamase primary sequence is shown. Above the sequence are the different amino acids that were identified at that sequence position among functional random mutants. Below the TEM-1 primary sequence are the different amino acids that appear at these positions in an alignment of 20 class A P-lactamases (Figure adapted from Huang et al., 1996).

Ml. Mabilat, C, and Courvalin, P., Development of Oligotyping for characterization and molecular epidemiology of TEM p-lactamases in Enterobacteriaceae. Antimicrob. Agents Chemo-ther. 34, AAC 230-290 (1990). 

The answer may be in discovering more specific mechanism-based P-lactamase inhibitors. It is curious that the best drugs developed so far are also P-lactam compounds (Fig. 6-14). One very interesting inhibitor is penicillanic acid sulfone (sulbactam, Fig. 6-14). This drug acts as a substrate (becomes hydrolyzed) and as an irreversible inactivator of TEM-type P-lactamase (covalently binding to it via molecular cleavage). 

Pseudomonas aemffnosa, p lactam resistance due to chromosomal p-lactamase derepression in Entercdmaer cloacae or plasmid-encoded TEM P-lacramase in Esc/ietichia coti, and tetracycline eliHux in E. coii, have no effect on the MIC of cationic peptides (66). Furthermore, they themselves do not tend to select resistant mutants, although some bacteria, such as Burldiolieria cepacia tend to be naturally resistant. 

P-Lactamases were first described in Staphylococcus aureus as factors causing resistance to penicillin (Kirby 1944). Later, the first plasmid-encoded P-lactamase, designated TEM based on the patient s name, was described in Greece from a strain of E. coli (Datta and Kontomichalou 1965). Currently, hundreds of different types of p-lactamases have been described. These enzymes are categorized based on their molecular properties (Ambler classification) or their hydrolytic pattern (Bush classification)—see Table 12.1 (Bush et al. 1995 Bush and Jacoby 2010). 

Farad WS, Pratt RF. Elimination of a good leaving group from the 3 -position of a cephalosporin need not be concerted with P-lactam ring opening TEM-2 P-lactamase-catalysed hydrolysis of pyridine-2-azo-4 -(N, N -dimethylanUine) cephalosporin (PADAC) and of cephaloridine. 1 Am Chem Soc. 1984 106 1489-90. 

From structure-activity correlations, it has been suggested that stability to p-lactamase action is conferred through steric hindrance by the 7a-methoxyl group at the enzyme active site (Birnbaum et al., 1978). Introduction of a 7a-methoxyl group depresses the rate of enzymatic hydrolysis by E. coll TEM -lactamase by a factor of 3 x 10" for cefoxitin relative to cephalothin (Fisher and Knowles, 1978). However, work from Knowles group suggests that the effects of the methoxyl substituent extend beyond a simple reduction in as the characteristics of the hydrolytic pathway are changed (see Fisher and Knowles, 1978). 

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