ACV synthetase forms the tripeptide precursor of penicillins and cephalosporins from alpha-aminoadipate, cysteine, and valine. Catalytic sites for substrate carboxyl activation as adenylates, peptide bond formations, epimerization and release of the tripeptide-thioester are integrated in multifunctional enzymes of 405 to 425 kD. These have been characterized from several pro- and eukaryotic beta-lactam producers. Implications of these results for the thio-template mechanism of peptide formation are discussed, as well as the use of this multienzyme as a model system for enzymatic peptide synthesis.

&(L-a-Aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase, the multienzyme catalyzing the formation of ACV from the constituent amino acids and ATP in the presence of Mg2+ and dithioerythritol, was purified about 2700-fold from Streptomyces clavuligerus. The molecular mass of the native enzyme as determined by gel filtration chromatography is 560 kDa, while that determined by denaturing gel electrophoresis is 500 kDa. The enzyme is able to catalyze pyrophosphate exchange in dependence on L-cysteine and L-valine, but no L-a-aminoadipic-acid-dependent ATP/PPi exchange could he detected. Other L-cysteine-and L-valine-activating enzymes present in crude extracts were identified as aminoacyl-tRNA synthetases which could be separated from ACV synthetase. The molecular mass of these enzymes is 140 kDa for L-valine ligase and 50 kDa for L-cysteine ligase. The dissociation constants have been estimated, assuming three independent activation sites, to be 1.25 mM and 1.5 mM for cysteine and ATP, and 2.4 mM and 0.25 mM for valine and ATP, respectively. The enzyme forms a thioester with a-aminoadipic acid and with valine in a molar ratio of 0.6: 1 (amino acid/enzyme). Thus, the bacterial ACV synthetase is a multifunctional peptide synthetase, differing from fungal ACV synthetases in its mechanism of activation of the non-protein amino acid.Up to this time, the family of penicillin and cephalosporin antibiotics have been the most important therapeutic agents to fight against infectious diseases in humans. Commercially, they are produced mainly by filamentous fungi, but it has been shown that the capacity to produce them is widespread in nature. The number of prokaryotic and eukaryotic microorganisms able to synthesize p-lactam antibiotics is continuously increasing [I]. The biosynthesis of penicillins and cephalosporins has been studied extensively, both biochemically and genetically [2,3]. Most of the enzymes involved were characterized and many of the genes encoding them have been cloned. The two initial steps of their biosynthesis were shown to be common to all penicillin-and cephalosporin-producing organisms studied so far. In the first step of the pathway, The formation of the tripeptide ACV by ACV synthetase has recently attracted the attention of many research groups. Banko et al. [4] were the first to suggest that ACV synthesis in Acremonium chrysogenum involves the action of a single multifunctional enzyme. Jensen et al. [5] have conducted similar studies using the P-lactam-producing organism Streptomyces clavuligerus and reported a cell-free system capable of synthesizing ACV and various structural analogs from the appropriate component amino acids. Recently, van Liempt et al. [6,7] have described the biosynthesis of ACV in A . niduluns in a reaction catalyzed by a multifunctional enzyme. The A . nidulans enzyme was purified 118-fold and was shown to catalyze ATP-PPi exchange in dependence of all three constituent amino acids, and the enzyme could be aminoacylated with ~-['~C]valine. It was suggested that the AC...

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For immunoelectron microscopic localization of isopenicillin N synthase (IPNS), glutaraldehyde-fixed mycelium of Penicillium chrysogenum PQ-96 was dehydrated by progressive lowering of the temperature and embedded into Lowicryl K4M at -3 5°C . This procedure resulted in good structural preservation such that the method of on-section labelling using antibody to IPNS from Cephalosporium acremonium CO 278 with the indirect antibody-gold technique could be successfully applied. By this method IPNS was localized in vesicular compartments belonging probably to the Golgi body and in the cell wall.

The occurrence, localization, and extraction of isopenicillin N-synthase (IPNS) were investigated in the gram-negative low-level 13-lactam producer Flavobacterium sp. strain SC 12.154, which forms deacetoxycephalosporin and excretes the cephabacin 7-formamidocephalosporin. IPNS was detected with anti-IPNS antibodies raised against the Cephalosporium acremonium enzyme. The flavobacterium enzyme, whose molecular mass (38 kilodaltons) and cofactor requirements resemble those of the fungal and Streptomyces enzymes, is formed at the transition from growth to the stationary phase. It was extracted into the polyethylene glycol phase of a polyethylene glycol-Ficoll-dextran three-phase system and was purified by quaternary aminoethyl ion-exchange chromatography, gel filtration, covalent chromatography on cystamine-Sepharose, and fast-protein liquid chromatography on Mono Q. The enzyme was characterized with respect to sulfhydryl requirement, inhibition by disulfides and metal ions, pH and temperature dependence, and stimulation by polyethylene glycol and low Triton X-100 concentrations, as well as by several amino acids, including a-aminoadipic acid and cysteine. The Km for a-aminoadipyl-cysteinyl-D-valine was 0.08 mM. An inactive membrane-associated form of IPNS was detected together with a P-lactamase active on isopenicillin N. The system has been suggested as a model for the study of endogenous functions of t3-lactams in bacteria.