On the two components of pyridoxal 5'-phosphate synthase from Bacillus subtilis.

Vitamin B6 is an essential nutrient in the human diet. It can act as a co-enzyme for numerous metabolic enzymes and has recently been shown to be a potent antioxidant. Plants and microorganisms have the ability to make the compound. Yet, studies of vitamin B6 biosynthesis have been mainly restricted to Escherichia coli, where the vitamin is synthesized from 1-deoxy-d -xylulose 5-phosphate and 4-phosphohydroxy-l-threonine. Recently, a novel pathway for its synthesis has been discovered, involving two genes (PDX1 and PDX2) neither of which is homologous to any of those participating in the E. coli pathway. In Bacillus subtilis, YaaD and YaaE represent the PDX1 and PDX2 homolog, respectively. The two proteins form a complex that functions as a glutamine amidotransferase, with YaaE as the glutaminase domain and YaaD as the acceptor and pyridoxal 5'-phosphate (PLP) synthesis domain. In this report we corroborate a recent report on the identification of the substrates of YaaD and provide unequivocal proof of the identity of the reaction product. We show that both the glutaminase and synthase reactions are dependent on the respective protein partner. The synthase reaction can also utilize an external ammonium source but, in contrast to other glutamine amidotransferases, is dependent on YaaE under certain conditions. Furthermore, we report on the detailed characterization of the inhibition of the glutaminase domain, and thus PLP synthesis, by the glutamine analog acivicin. Employing pull-out assays and native-PAGE, we provide evidence for the dissociation of the bi-enzyme complex under these conditions. The results are discussed in light of the nature of the interaction of the two components of the enzyme complex.

A G-box element from the Catharanthus roseus strictosidine synthase (Str) gene promoter confers seed-specific expression in transgenic tobacco plants.

The enzyme encoded by the strictosidine synthase (Str) gene from Catharanthus roseus catalyses a key step in the biosynthesis of the pharmaceutically important terpenoid indole alkaloids. Str cDNA and genomic clones have already been isolated, allowing us to study the regulation of Str gene expression. Here we focus on the role of a putative cis-acting element, CACGTG, in the Str promoter. This sequence is known as a G-box, and functions as a transcription-regulating sequence in a number of other promoters. By means of electrophoretic mobility shift assays it was demonstrated that the Str G-box is capable of interacting with nuclear factors in tobacco and with the cloned tobacco G-box-binding factor TAF-1. Disruption of the Str G-box sequence by two single-nucleotide mutations prevented binding of factors, thereby demonstrating the specificity of the observed interactions. Functional analysis in transgenic tobacco plants demonstrated that these mutations also reduced the transcriptional activity of constructs containing tetramers of the Str G-box sequence. Expression directed by a tetramer of the Str G-box fused to a truncated promoter containing only a TATA box was confined to seeds and was found to increase during seed maturation. Thus, the Str G-box tetramer is able to direct seed-specific expression independently of other regulatory sequences. G-box-directed expression in leaves required the presence of an enhancer region from the cauliflower mosaic virus (CaMV) 35S promoter. The results indicate that the G-box needs to interact with other elements to drive expression in leaf, and that it can by itself confer seed-specific expression as a multimer. The fact that only some of the G-boxes found in different promoters serve as seed-specific elements indicates that sequences flanking the G-box determine the transcriptional activity in different tissues. Based on sequence comparisons we propose that the nucleotides at positions -4, -3, -2 and/or +4 are important in determining seed-specific expression.

The genomic organization of Isopeptidase T-3 (ISOT-3), a new member of the ubiquitin specific protease family (UBP).

A novel Isopeptidase T gene (ISOT-3) has been identified on human mosome 3q26.2--q26.3. gene shows 67.3% nucleotide identity and 54.8% amino acid identity to n Isopeptidase (ISOT-1). Northern blot analysis has shown that ISOT-3 is highly essed in ovary and testes, low-level expression in six other tissues tested. In contrast, ISOT-1 is essed at high levels in brain, and there is no detectable expression in ovary. The exonic nization of these two genes highly conserved with only one variant intron position. Intron 15 in -3 is absent in ISOT-1, there is an alternate splice site at the same location. Although the --intron structure has been erved between the two genes, ISOT-3 has significantly larger intronic ons, and the overall of this gene is at least 90 kb compared to 15 kb for ISOT-1. These data suggest that both ISOT-1 and ISOT-3 have descended from a common ancestor. In addition, the low overall sequence identity and different expression patterns may reflect differences in substrate specificity.

Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus.

Cells of Catharanthus roseus (L.) G. Don were genetically engineered to over-express the enzymes strictosidine synthase (STR; EC 4.3.3.2) and tryptophan decarboxylase (TDC; EC 4.1.1.28), which catalyze key steps in the biosynthesis of terpenoid indole alkaloids (TIAs). The cultures established after Agrobacterium-mediated transformation showed wide phenotypic diversity, reflecting the complexity of the biosynthetic pathway. Cultures transgenic for Str consistently showed tenfold higher STR activity than wild-type cultures, which favored biosynthetic activity through the pathway. Two such lines accumulated over 200 mg.L-1 of the glucoalkaloid strictosidine and/or strictosidine-derived TIAs, including ajmalicine, catharanthine, serpentine, and tabersonine, while maintaining wild-type levels of TDC activity. Alkaloid accumulation by highly productive transgenic lines showed considerable instability and was strongly influenced by culture conditions, such as the hormonal composition of the medium and the availability of precursors. High transgene-encoded TDC activity was not only unnecessary for increased productivity, but also detrimental to the normal growth of the cultures. In contrast, high STR activity was tolerated by the cultures and appeared to be necessary, albeit not sufficient, to sustain high rates of alkaloid biosynthesis. We conclude that constitutive over-expression of Str is highly desirable for increased TIA production. However, given its complexity, limited intervention in the TIA pathway will yield positive results only in the presence of a favorable epigenetic environment.