Tryptic peptides of the novel ceftazidimase CAZ-5 were sequenced by manual Edman degradation and aligned according to strong homology (more than 98%) with SHV-1 and SHV-2 13-lactamase sequences. differed from SHV-1 by five amino acid substitutions. Unusually high activity of CAZ-5 towards ceftazidime was imputed to substitution of a Lys for a Glu at position 214 of the mature protein.Broad-spectrum cephalosporins such as cefotaxime and related compounds, which are very effective against gramnegative bacteria, have been extensively used in hospital practice. However, infections caused by members of the family Enterobacteriaceae resistant to newer cephalosporins were soon observed (23,24). In part, the resistance was related to overproduction of chromosomally mediated cephalosporinase involving a stable derepression of enzyme synthesis (17,20). Otherwise, transferable resistance to broad-spectrum cephalosporins, first detected in the Federal Republic of Germany (15, 16), was responsible, especially in France, for outbreaks of nosocomial infections (6,14,26). Whatever the main bacterial phenotype (cefotaxime resistance, ceftazidime resistance, or multiresistance), it was associated with the synthesis of new plasmid-mediatedlactamases (5,7,8,10,12,21,25).Recently, such as enzyme, CAZ-5, also called SHV-4 (22), was isolated (Hopital Begin, St. Mandd, France) from Klebsiella pneumoniae 210-2, a strain resistant to broad-spectrum cephalosporins. The kinetic constants of CAZ-5 did not notably differ from those of the cefotaxime-hydrolyzing ,B-lactamase SHV-2, with the exception of the hydrolysis rates for ceftazidime and aztreonam, which were markedly increased ( markedly from that of the SHV-1 protein (3) and was in good agreement with that deduced from the amino acid sequence analysis. Eight first N-terminal sequence analyses each yielded one residue with the sequence Ser-Pro-Gln-ProLeu-Glu-Gln-Ile-identical to those of SHV-1 and SHV-2.The S-carboxymethylated protein was digested by trypsin (Sigma Chemical Co.), and the resulting peptide mixture was resolved by high-performance liquid chromatography on a Waters ,uBondapak C18 column (10 pum; 4 by 300 mm) (Fig. 1). Elution was performed with 0.1% trifluoroacetic acid in water as solvent A and acetonitrile as solvent B. A threestep linear gradient of solvent B was used (0 min, 5%; 35 min, 30%; 45 min, 45%; 60 min, 80%) with a flow rate of 1 ml/min. Apart from peak 16, which exhibited a higher hydrophobicity, the elution pattern of the tryptic peptides of CAZ-5 was identical to that of SHV-2 (2). Peaks 11 to 14, 3, and 5 to 22 were further chromatographed at pH 6.0 on the same column, with a linear gradient from 0.25 mM ammonium acetate buffer to 0.5 mM ammonium acetate bufferacetonitrile (2:3, vol/vol). Peptide T-19, which eluted with
