Citrohacter freundii GN346 produces a class C p-lactamase exhibiting the substrate profile of a typical cephalosporinase. The structural and promoter regions of the cephalosporinase gene, comprising 1408 nucleotides, were completely sequenced. The amino acid sequence of the mature enzyme, comprising 361 amino acids, and its molecular mass, 39 878 Da, were determined. The active site was confirmed to be Ser-64. The amino acid sequence of the enzyme differs from that of the cephalosporinase of C. jreundii OS60 by nine residues. The nucleotide sequence of the promoter region suggests a possible attenuator structure. Lys-67, one of the most conserved residues found in class A and C p-lactamases and penicillin-binding proteins, was converted into arginine, threonine or glutamic acid through site-directed mutagenesis. The Glu-67 enzyme had lost the catalytic activity and the Thr-67 enzyme only showed a trace of activity. The Arg-67 enzyme, which retained a significant amount of the activity, was purified. The K, values of the Arg-67 enzyme for cephalothin, cephaloridine and benzylpenicillin are 13-19 times those of the wild-type enzyme; the k,,, values for the three substrates are 37%, 3%, and 36% those of the wild-type enzyme, respectivelyThe cephalosporinases found in a large number of Gramnegative bacteria are species-specific p-lactamases [I, 21. These p-lactamases belong to class C, according to Ambler's classification based on the amino acid sequence around the active site [3]. In the clinical field, cephalosporinases are of importance because clinical isolates producing cephalosporinases show high levels of resistance to both cephalosporins and penicillins [4]. In the preceding studies [2, 5-71, we selected the cephalosporinase of Citrobucter,freundii GN346 as a representative cephalosporinase of Gram-negative bacteria and its characteristics were extensively investigated. C. freundii GN346 was isolated from a clinical source in Japan in 1965, and produces a chromosomal p-lactamase exhibiting the substrate profile of a typical cephalosporinase [8]. The cephalosporinase is synthesized through a unique regulatory mechanism of thermolabile repression [9]. It is synthesized semiconstitutively when the bacterium is cultured at above 25"C, whereas the enzyme synthesis requires a chemical inducer such as benzylpenicillin at 20 "C. The cephalosporinase gene of GN346 was cloned into a vector plasmid and its expression in Escherichia coli was confirmed [lo]. Recently, the structural gene for the enzyme was sequenced and a preliminary report of the amino acid sequence of the mature enzyme has been presented at a symposium [7].

[reaction: see text] An intermolecular aza-double Michael reaction leading to functionalized piperidin-2-ones from simple starting materials has been developed. The method allows alpha,beta-unsaturated amides to be used as a synthon of the piperidine nucleus. In addition, the utility of this methodology is demonstrated by its application to a formal synthesis of the indolo[2,3-a]quinolizidine alkaloids, (+/-)-deplancheine, (+/-)-tacamonine, and the antidepressant paroxetine.

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The conserved KTG triad in the class C /I-lactamase from Cirrohucter,freundii GN346 was examined as to its function by means of site-directed mutagencsls.The following conversions were performed; Lys-3 15 to arginine, alanine or glutamic acid, Thr-3 I6 to valine, and Gly-3 I7 to alanine, proline or isoleucine. The resultant mutant enzymes revealed that a basic amino acid at position 315 and a small uncharged residue at position 317 are essential for the enzyme activity, but a hydroxyl group at residue 316 is not required for the enzymatic catalysis. The kinetic properties of the purified Arg-3 15 and Val-3 16 enzymes provided information on the function of these residues.