Histidinol phosphate phosphatase, catalyzing the penultimate step of the histidine biosynthesis pathway, is encoded by ytvP (hisJ) in Bacillus subtilis.
The deduced product of the Bacillus subtilis ytvP gene is similar to that of ORF13, a gene of unknown function in the Lactococcus lactis histidine biosynthesis operon. A B. subtilis ytvP mutant was auxotrophic for histidine. The only enzyme of the histidine biosynthesis pathway that remained uncharacterized in B. subtilis was histidinol phosphate phosphatase (HolPase), catalyzing the penultimate step of this pathway. HolPase activity could not be detected in crude extracts of the ytvP mutant, while purified glutathione S-transferase-YtvP fusion protein exhibited strong HolPase activity. These observations demonstrated that HolPase is encoded by ytvP in B. subtilis and led us to rename this gene hisJ. Together with the HolPase of Saccharomyces cerevisiae and the presumed HolPases of L. lactis and Schizosaccharomyces pombe, HisJ constitutes a family of related enzymes that are not homologous to the HolPases of Escherichia coli, Salmonella typhimurium, and Haemophilus influenzae. (+info)
Positive control of lac operon expression in vitro by guanosine 5'-diphosphate 3'-diphosphate.
Maximal expression of the Escherichia coli lactose operon in a coupled in vitro transcription-translation system from a Salmonella typhimurium relA mutant was strongly dependent upon addition of guanosine 5'-diphosphate 3'-diphosphate (ppGpp). Without added ppGpp, at saturating 3',5'-cyclic AMP (cAMP) concentrations, synthesis of beta-galactosidase (beta-D-galactoside galactohydrolase, EC 126.96.36.199) was reproducibly only 5-7% of that which can be obtained with 0.5-0.8 mM ppGpp. Experiments in which transcription was uncoupled from translation indicated that this 14- to 20-fold stimulation by ppGpp occurred at the level of transcription. When coupled beta-galactosidase synthesis was primed with a template containing a well-characterized mutant lac promoter (lacP(r)L8UV5), the dependence on ppGpp was greatly reduced. This result provides an important experimental control previously unavailable for verifying the significance of ppGpp effects on gene regulation in vitro; it indicates that activation of lacP(+) expression by ppGpp is specifically an effect of increased transcription initiations. Furthermore, the large ppGpp stimulation of lacP(+) DNA enabled the level of expression of this template to approach that of lacP(r)L8UV5 DNA, an observation expected from results in vivo but not obtained with other transcription-translation systems in vitro. The importance of these results is considered with respect to previous ideas on the physiological role of ppGpp as a supercontrol molecule in bacterial regulation. (+info)
Deletions fusing the hisG and hisD genes in Salmonella typhimurium.
Frameshift mutation hisD497 occurs in the operator-proximal portion of the Salmonella typhimurium gene coding for the dimeric protein, L-histidinol dehydrogenase (HDH). Rare revertants of hisD497 are deletions fusing the hisD gene to the adjacent preceding structural gene, hisG (adenosine 5'-triphosphate-PR transferase). HDH purified from one revertant, hisGD4908, contains subunits of approximately normal molecular weight but with no clearly demonstrable unique amino-terminal sequence. We propose that a combined inactive G-D polypeptide is synthesized and then cleaved at a number of closely juxtaposed sites by endoproteolytic activity. At least some of the resulting fragments then participate in formation of active HDH dimers. (+info)
Derepression and repression of the histidine operon: role of the feedback site of the first enzyme.
Thiazolealanine, a false feedback inhibitor, causes transient repression of the his operon previously derepressed by a severe histidine limitation in strains with a wild-type or feedback-hypersensitive first enzyme but not in feedback-resistant mutants. Since experiments reported here clearly demonstrate that thiazolealanine is not transferred to tRNAHis, it is proposed that this "transient repression" is effected through the interaction of thiazolealanine with the feedback site of the enzyme. Experiments in the presence of rifampin indicate that this thiazolealanine-mediated effect is exerted at the level of translation. We conclude that histidine (free), in addition to forming co-repressor, also represses the operon at the level of translation through feedback interaction with the first enzyme of the pathway (adenosine 5'-triphosphate phosphoribosyltransferase). Rates of derepression in feedback-resistant strains are roughly half of those observed in controls, suggesting a positive role played by a first enzyme with a normal but unoccupied feedback site. Some feedback-resistant mutants, in contrast to the wild type, were unable to exhibit derepression under histidine limitation caused by aminotriazole. (+info)
Regulation of macromolecular synthesis in reovirus-infected L-929 cells I. Effect of L-histidinol.
The histidine analogue L-histidinol, reported by Vaughan and Hansen (1973) to establish a potent, readily reversible inhibition of eukaryotic protein synthesis in vivo, was used to investigate the regulation of macromolecular synthesis in reovirus-infected L-929 cells. The addition of L-histidinol to normal L cells led to a total inhibition of protein synthesis. The inhibition appeared to be a consequence neither of isotope dilution resulting from elevated endogenous amino acids nor of an inability of treated cells to accumulate exogenous amino acids. Addition of L-histidine to histidinol-arrested cells resulted in a complete recovery of protein synthesis. Similarly, protein synthesis in reovirus-infected L cells examined 17 h postinfection (31 C) was totally inhibited by histidinol treatment and was readily reversed by the addition of histidine. Reovirus-infected cells treated with histidinol had an essentially unaltered capacity to synthesize reovirus single-stranded RNA relative to unperturbed cultures but a diminishing ability to maintain genome RNA synthesis. Addition of L-histidine to arrested cultures led to a complete recovery of genome RNA synthesis. The L-histidinol-mediated arrest of protein synthesis was both very effective and easily reversed, suggesting the general applicability of this novel inhibitor to investigations of regulation of macromolecular synthesis in both normal and virus-infected eukaryotic cells. (+info)
Studies on the role of uncharged tRNA in pleiotypic response of animal cells.
Experiments were carried out to assess the physiological significance of the charging level of tRNA. Histidinol, a competitve inhibitor of charging of tRNAHis, was used to induce uncharged tRNA in mammalian cells. It is demonstrated that both in the presence of histidinol and under histidine depletion about 40% of the tRNAHis is uncharged. Concomitant with this appearance of uncharged tRNA(a) the pools of GTP and ATP are decreased rapidly by 25--30%; (b) the synthesis of both protein and ribosomal RNA is inhibited, whereas that of nucleoplasmic RNA is not affected; (c) the uptake of 2-deoxyglucose, phosphate, Ca2+; uridine and adenosine is inhibited; and (d) the growth of 3T6 fibroblasts is arrested. It is suggested that the appearence of uncharged tRNA is one of the earliest events occurring under conditions of amino acid starvation, which in turn causes the various metabolic changes observed. (+info)
Analysis of an L-histidinol-utilizing mutant of Pseudomonas aeruginosa.
Transductional analysis was applied to the Pseudomonas aeruginosa mutant PAO14 (hnc-1). This mutant can utilize L-histidinol as sole source of carbon and nitrogen and has a 60-fold increased histidinol dehydrogenase (HDH) content (Dhawale, Creaser & Loper, 1972). Transductional analysis was carried out using 18 histidine-requiring mutants to see where the hnc-1 locus maps in relation to the structural genes of histidine biosynthesis. The hnc-1 marker cotransduced with group IV genes at 97 to 100 % and not at all with group I, which is known to be the structural gene for HDH. The data obtained in the studies of Km (histidinol) and Km (NAD), and the effect of pH and temperature on the HDH activity from PAO1 and PAO14 are in full agreement with the genetic data that the hnc-1 mutation is not in the structural gene for HDH. It is suggested that hnc-1 may be a mutation in a regulatory gene affecting HDH synthesis in PAO14 and may map close to his-IV whose function in histidine biosynthesis is not known. (+info)
Purification of histidase from Streptomyces griseus and nucleotide sequence of the hutH structural gene.
Histidine ammonia-lyase (histidase) was purified to homogeneity from vegetative mycelia of Streptomyces griseus. The enzyme was specific for L-histidine and showed no activity against the substrate analog, D-histidine. Histidinol phosphate was a potent competitive inhibitor. Histidase displayed saturation kinetics with no detectable sigmoidal response. Neither thiol reagents nor a variety of divalent cations had any effect on the activity of the purified enzyme. High concentrations of potassium cyanide inactivated histidase in the absence of its substrate or histidinol phosphate, suggesting that, as in other histidases, dehydroalanine plays an important role in catalysis. The N-terminal amino acid sequence of histidase was used to construct a mixed oligonucleotide probe to identify and clone the histidase structural gene, hutH, from genomic DNA of the wild-type strain of S. griseus. The cloned DNA restored the ability of a histidase structural gene mutant to grow on L-histidine as the sole nitrogen source. The deduced amino acid sequence of hutH shows significant relatedness with histidase from bacteria and a mammal as well as phenylalanine ammonia-lyase from plants and fungi. (+info)