A 1536-nucleotide-long sequence that carries the ampC fi-lactamase gene of the Escherichia coli K-12 chromosome has been determined. This gene codes for a protein of 377 amino acids, of which the first 19 amino acids form a signal peptide. The molecular weight of the mature enzyme was determined to be 39,600. The ampC P-lactamase with a substrate specificity for cephalosporins showed no significant sequence homologies with (-lactamases of the penicillinase type or with D-alanine carboxypeptidases. However, because the region around serine-80 of the ampC f3-lactamase has extensive homology with an active-site fragment ofthe Pseudomonas aeruginosa cephalosporinase, we suggest that the ampC cephalosporinase as well as related cephalosporinases form a distinct group of serine P-lactamases that have an evolutionary origin different from that of the serine penicillinases and thus constitute a new class of 13-lactamases. f3Lactamases of chromosomal or plasmid origin have been found in a large number of Gram-positive and Gram-negative bacteria (1). These enzymes have been classified according to such properties as substrate profile, isoelectric point, and molecular weight. The protein sequence has been determined for ,-lactamases from Staphylococcus aureus PC1 (2), Bacillus ltcheniformis 749/C (3, 4), and Escherichia coli/R6K, R-TEM (5).Nearly the entire sequence ofthe B. cereus 569/H P-lactamase has also been elucidated (6, 7). In addition, the amino acid sequence of the ,B-lactamase encoded by the plasmid pBR322 has been deduced from its nucleotide sequence (8). The two plasmid-mediated TEM P-lactamases from Gram-negative species differ only by one amino acid residue (5, 8).These 3-lactamases of known sequence all show substrate specificity for penicillins and have therefore been termed "penicillinases" (1). The molecular weight ofthese enzymes is around 29,000 (7). They show significant sequence homologies with each other (7) as well as with regions of D-alanine carboxypeptidases from B. stearothermophilus and B. subtilis (9). By the use of substrate analogues, the active site has been determined for three penicillinases and two carboxypeptidases (9-13). They have been referred to as "serine enzymes" because the reagents react with a serine residue. ,3-Lactamases ofthe metalloenzyme type have also been identified. From incomplete sequence data it is suggested that this class has a different evolutionary origin from that of the serine penicillinases of known sequence (7).The chromosomally encoded ,B-lactamases of Gram-negative enterobacteria in general are basic proteins with a substrate specificity for cephalosporins (14). One such cephalosporinase is encoded by the ampC gene of E. coli K-12. This gene, which is located at 93.8 min on the E. coli linkage map (15), was isolated from a gene bank containing E. coli chromosomal DNA (16,17). By subcloning, ampC was localized to a 1370-base-pair (bp)-long DNA fragment (17, 18). We have reported (19) a characterization of the regulatory region that precedes ampC. T...