Identification of Amino Acid Substitutions That Alter the Substrate Specificity of TEM - 1 1 - Lactamase TIMOTHY

TEM-1 1-lactamase is the most prevalent plasmid-mediated 13-lactamase in gram-negative bacteria. Recently, TEM I-lactamase variants with amino acid substitutions in the active-site pocket of the enzyme have been identified in natural isolates with increased resistance to extended-spectrum cephalosporins. To identify other amino acid substitutions that alter the activity of TEM-1 towards extended-spectrum cephalosporins, we probed regions around the active-site pocket by random-replacement mutagenesis. This mutagenesis technique involves randomizing the DNA sequence of three to six codons in the blaTEM-l gene to form a library containing all or nearly all of the possible substitutions for the region randomized. In total, 20 different residue positions that had been randomized were screened for amino acid substitutions that increased enzyme activity towards the extended-spectrum cephalosporin cefotaxime. Substitutions at positions 104, 168, and 238 in the TEM-1 P-lactamase that resulted in increased enzyme activity towards extended-spectrum cephalosporins were found. In addition, small deletions in the loop containing residues 166 to 170 drastically altered the substrate specificity of the enzyme by increasing activity towards extended-spectrum cephalosporins while virtually eliminating activity towards ampicillin. P-Lactam antibiotics such as penicillins and cephalospo-rins are among the most often used antimicrobial agents. Because of the selective pressure resulting from the prevalence of j-lactam antibiotic use, bacterial resistance has increased and now represents a serious threat to antibiotic therapy (14, 19). The production of ,B-lactamase enzymes that cleave the amide bond in the ,-lactam ring to generate inactive products is the most common mechanism of bacterial resistance to ,B-lactam antibiotics (27). Genes that encode ,B-lactamases can be found on the bacterial chromosome or on plasmids (26, 39). Expression of chromosomal enzymes is often inducible, with ,B-lactam antibiotics serving as the inducers (39). Plasmid-mediated enzymes are commonly expressed constitutively (27). Plas-mid-mediated enzymes are a particular concern because they can be transferred to distantly related bacteria by conjugation or transduction. On the basis of primary sequence homology, f-lactamases have been grouped into four classes (A, B, C, and D) (2). Classes A, C, and D involve a serine residue at the active site (7, 16). Class B enzymes are less abundant and require a catalytic zinc for activity (45). The active-site serine ,-lactamases belong to a larger family of penicillin-recognizing enzymes that includes the penicillin binding proteins that are involved in cell wall biosynthe-sis and are the lethal targets of 3-lactam antibiotics (20). All of these enzymes contain the active-site serine and a conserved …