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An 11450-base DNA fragment containing the gene for the extracellular active-site serine DD-peptidase of Streptornyces R61 was cloned in Streptornyces lividans using the high-copy-number plasmid pi J702 as vector. Amplified expression of the excreted enzyme was observed. Producing clones were identified with the help of a specific antiserum directed against the pure DD-peptidase. The coding sequence of the gene was then located by hybridization with a specific nucleotide probe and sub-fragments were obtained from which the nucleotide sequence of the structural gene and the putative promoter and terminator regions were determined. The sequence suggests that the gene codes for a 406-amino-acid protein precursor. When compared with the excreted, mature DDpeptidase, this precursor possesses a cleavable 31-amino-acid N-terminal extension which has the characteristics of a signal peptide, and a cleavable 26-amino-acid C-terminal extension. On the basis of the data of Joris et al. (following paper in this journal), the open reading frame coding for the synthesis of the DD-peptidase was established. Comparison of the primary structure of the Streptomyces R61 DD-peptidase with those of several active-site serine β-lactamases and penicillin-binding proteins of Escherichia coli shows homology in those sequences that comprise the active-site serine residue. When the comparison is broadened to the complete amino acid sequences, significant homology is observed only for the pair Streptomyces R61 DD-peptidase/Escherichia coil ampC β-lactamase (class C). Since the Streptomyces R61 DD-peptidase and β-lactamases of class A have very similar three-dimensional structures [Kelly et al. (1986) Science (Wash. DC) 231, 1429-1431; Samraoui et al. (1986) Nature (Lond.) 320, 378-380], it is concluded that these tertiary features are probably also shared by the β-1actamases of class C, i.e. that the Streptomyces R61 DD-peptidase and the β-lactamases of classes A and C are related in an evolutionary sense. [ABSTRACT FROM AUTHOR]

The cephalosporinase of Citrobacter freundii GN346 is a class-C β-1actamase comprising 361 amino acids. The substitution of the glutamic acid at position 219 in the enzyme by lysine was previously shown to broaden its substrate specificity to unfavorable substrates such as oxyimino cephalosporins [Tsukamoto, K., Ohno, R. & Sawai, T. (1990) J. Bacteriol. 172, 4348–4351]. To investigate the cause of this phenomenon, Glu219 was changed to glutamine, cysteine or tryptophan. All the resultant enzymes showed higher cefuroxime-hydrolytic activities than the wild type, the order of increasing cefuroxime-hydrolytic activity being as follows: Trp>Lys>Cys>Gln>Glu. The rate of hydrolysis of cefuroxime by the Trp219 enzyme was approximately 3×104 times that of the wild- type eo.zyme. The order of increasing cefuroxime hydrolysis was approximately proportional to the molecular volume of the amino acid substituted and independent of the ionic character of the amino acids. The cysteine residue at position 219 in the Cys219 enzyme allowed its complete reaction with an SH-blocking reagent, 4-chloromercuriphenylsulfonic acid. The modified enzyme with the bulkier residue showed a 45% higher cefuroxime-hydrolytic activity than the untreated enzyme. These results suggested that extension of the substrate spectrum may be attributed to alteration in the configuration of the enzyme around position 219. [ABSTRACT FROM AUTHOR]