The two genes, nadA and nadB, responsible for quinolinate biosynthesis from aspartate and dihydroxyacetone phosphate in Escherichia coli were cloned and characterized. Quinolinate (pyridine-2,3-dicarboxylate) is the biosynthetic precursor of the pyridine ring of NAD.Gene nadA was identified by complementation in three different nadA mutant strains. Sequence analysis provided an 840-bp open reading frame coding for a 31 555-Da protein. Gene nadB was identified by complementation in a nadB mutant strain and by the L-aspartate oxidase activity of its gene product. Sequence analysis showed a 1620-bp open reading frame coding for a 60306-Da protein.For both genes, promoter regions and ribosomal binding sites were assigned by comparison to consensus sequences. The nadB gene product, L-aspartate oxidase, was purified to homogeneity and the N-terminal sequence of 19 amino acids was determined. The enzyme was shown to be specific for L-aspartate.High-copy-number vectors, carrying either gene nadA, nadB or nadA + nadB, increased quinolinate production 1.5-fold, 2.0-fold and 15-fold respectively. Both gene products seem to be equally rate-limiting in quinolinate synthesis.Despite the central importance of NAD to metabolism, the biochemistry and regulation of its de novo formation are still not completely clarified. In all known organisms examined to date the pyridine ring of the NAD molecule is synthesized via quinolinate (pyridine-2,3-dicarboxylate) ; quinolinate formation occurs via four different pathways depending on the organism [l -31.In Escherichia coli and Salmonella typhimurium quinolinate biosynthesis was suggested to be catalyzed by a quinolinate synthetase complex [4] from L-aspartate and dihydroxyacetone phosphate [2, 31. Up to six intermediates have been postulated for the conversion of L-aspartate and dihydroxyacetone phosphate into quinolinate [2], an exact reaction sequence, however, has not been established yet.The quinolinate synthetase complex consists of the two enzymes quinolinate synthetases A and B, which are the gene products of genes nadA and nadB. Quinolinate synthetase B has been identified and characterized as FAD-dependent L-aspartate oxidase [5]. It catalyses oxidation of L-aspartate to iminoaspartate which is condensed with dihydroxyacetone phosphate to quinolinate under the action of quinolinate synthetase A (Fig. 1). Correspondence to H. G. Quinolinate is further converted to nicotinic acid mononucleotide via decarboxylation and phosphoribosylation by the nadC gene product quinolinate phosphoribosyltransferase [6] (Fig. 1). nadC mutant strains are not able to convert and thus excrete quinolinate into the medium.The three structural genes, nadA, nadB and nadC have been located at 17 min, 56 min [7] and 1.5 min [8], respectively, on the E. coli genetic linkage map [9]; very similar locations (17 min, 55 min and 3 min respectively) were identified [lo] on the S. typhimurium genetic linkage map.In order to investigate the molecular biology of quinolinate biosynthesis we have cloned and sequen...

We have examined the biochemical degradation of an isolated basement membrane matrix (bovine lens capsule) by human liver cathepsins B, H and L and the cathepsin B-like proteinase from malignant ascitic fluid. This study was carried out using two different methods: The first strategy was to follow the liberation of soluble proteins and peptides as a function of time at different pHs. Then the digestion products were characterized, as collagen IV, fibronectin and laminin fragments, using monospecific polyclonal antibodies and a quantitative dot-blot analysis. From these results, the ability of the four proteinases to digest "in vitro" intact bovine lens capsule in the physiological pH range is demonstrated. Cathepsin L is the most powerful against the three membrane components studied. As shown by electroelution and immunochemical quantitation, the digestion would be a consequence of proteinases binding to the capsule. With intact basement membrane as a substrate a "in vitro" molecular analysis of this digestion process was possible by these methods. On this basis, the "in vivo" secretion of cysteine proteinases during malignancy would be related to the local basement membrane dissolution associated with tumor invasion.

To investigate the degree of similarity between picornavirus proteases, we cloned the genomic cDNAs of an enterovirus, echovirus 9 (strain Barty), and two rhinoviruses, serotypes 1A and 14LP, and determined the nucleotide sequence of the region which, by analogy to poliovirus, encodes the protease. The nucleotide sequence of the region encoding the genome-linked protein VPg, immediately adjacent to the protease, was also determined. Comparison of nucleotide and deduced amino acid sequences with other available picornavirus sequences showed remarkable homology in proteases and among VPgs. Three highly conserved peptide regions were identified in the protease; one of these is specific for human picornaviruses and has no obvious counterpart in encephalomyocarditis virus, foot-and-mouth disease virus, or cowpea mosaic virus proteases. Within the other two peptide regions two conserved amino acids, Cys 147 and His 161, could be the reactive residues of the active site. We used a statistical method to predict certain features of the secondary structures, such as alpha helices, beta sheets, and turns, and found many of these conformations to be conserved. The hydropathy profiles of the compared proteases were also strikingly similar. Thus, the proteases of human picornaviruses very probably have a similar three-dimensional structure.