Histidinol Dehydrogenase Activity Research Articles

Anti-virulence Strategy against Brucella suis: Synthesis, Biological Evaluation and Molecular Modeling of Selective Histidinol Dehydrogenase Inhibitors

In the facultative intracellular pathogen Brucella suis, histidinol dehydrogenase (HDH) activity, catalyzing the last step in histidine biosynthesis, is essential for intramacrophagic replication. The inhibition of this virulence factor by substituted benzylic ketones was a proof of concept that disarming bacteria leads to inhibition of intracellular bacterial growth in macrophage infection. This work describes the design, synthesis and evaluation of 19 new potential HDH inhibitors, using a combination of classical approaches and docking studies. The IC(50)-values of these inhibitors on HDH activity were in the nanomolar range, and several of them showed a 70-100% inhibition of Brucella growth in minimal medium. One selected compound yielded a strong inhibitory effect on intracellular replication of B. suis in human macrophages at concentrations as low as 5 μM, with an overall survival of intramacrophagic bacteria reduced by a factor 10(3). Docking studies with two inhibitors showed a good fitting in the catalytic pocket and also interaction with the second lipophilic pocket binding the cofactor NAD(+). Experimental data confirmed competition between inhibitors and NAD(+) at this site. Hence, these inhibitors can be considered as promising tools in the development of novel anti-virulence drugs.

Method for determination of histidine in tissues by isocratic high-performance liquid chromatography and its application to the measurement of histidinol dehydrogenase activity in six cattle organs

A selective and simple HPLC procedure has been developed to determine histidine (His) and histidinol (HDL) in liver supernate. The separation was performed on a column, Mightysil RP-18 GP. The eluted analytes were measured with UV detection without derivatization which provided detection limits of 1.1 and 2.0 μ M for His and HDL ( S/ N ratio, 3:1), respectively. Recovery of the analytes added to liver sample was 98.3–101.6% within a 1-day study and 95.7–98.6% on different day (6 days) studies. The apparent histidinol dehydrogenase activities (nmol/g wet tissue) at pH 8, 9, 10, 11, and 12 were 38.6, 50.4, 160.3, 274.3, and 185.6 for liver; 90.6, 132.2, 30.7, 22.1, and 6.76 for kidney; 0.0, 0.0, 38.2, 20.1, and 12.9 for pancreas; 0.0, 0.0, 0.0, 14.7, and 6.8 for spleen; 0.0, 0.0, 4.2, 6.8, and 0.0 for muscle; and 0.0, 0.0, 4.9, 1.8, and 0.0 for small intestine, respectively. On the basis of optimum pH values, histidinol dehydrogenase activity in the organs was in the following order: liver>kidney>pancreas>spleen>muscle>small intestine.

New vectors in fission yeast: application for cloning the his2 gene

We describe a new Escherichia coli vector (pON5) that allows positive selection for recombinant clones. In this plasmid, the bla gene from pBR322 is permanently active, whereas the neo gene from transposon Tn5 is repressed by the cI-encoded λ. repressor. When DNA is inserted into the Bc/I or HindIII restriction sites situated within the cI gene, the neo gene becomes transcribed from the λ P R promoter. We have also made a Schizosaccharomyces pombe derivative of pON5 (= pON163) by introducing the fission yeast arsl and ura4 + sequences. We show that this plasmid is capable of transforming Sc. pombe ura4 strains, as well as ura3 strains of the distantly related budding yeast Saccharomyces cerevisiae. We have used pON163 for the construction of two fission yeast genomic libraries. From these gene banks clones were isolated that were able to complement fission yeast his2 mutants. Such plasmids could also rescue his4C mutants of Sa. cerevisiae, defective in the histidinol dehydrogenase activity of the multifunctional HIS4 gene product. Finally, we describe the plasmid pDW232 which is useful for functional analysis of fission yeast genes. It is a pGEM3 derivative adapted to fission yeast, carrying multiple cloning sites between the T7 and SP6 promoters, together with arsl and ura4 + from Sc. pombe.

Isolation of Candida tropicalis auxotrophic mutants

An enrichment scheme using nystatin was designed for the isolation of auxotrophic mutants from the diploid-alkane-utilizing yeast Candida tropicalis. A collection of 194 auxotrophs representing 7 phenotypes was isolated. One class of mutants was identified as having a defect in histidinol dehydrogenase activity and a second class of mutants was identified as having a defect in orotidine monophosphate decarboxylase activity. These strains are good candidates to be carrying mutations corresponding to the HIS4 and URA3 genes of Saccharomyces cerevisiae.

Isolation of the Candida albicans histidinol dehydrogenase (HIS4) gene and characterization of a histidine auxotroph

Genetic studies were done with Candida albicans CBS 562. Various auxotrophs were isolated following mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. SAG5 (his4C), a stable histidine auxotroph defective in histidinol dehydrogenase activity, was characterized and chosen for further molecular studies. Therefore, the C. albicans HIS4 gene was isolated. The gene was obtained from a genomic library of the wild-type strain, which was constructed in plasmid YEp24. The HIS4 gene was isolated by transformation of a Saccharomyces cerevisiae strain that carried a his4 mutation. The isolated C. albicans HIS4 gene complemented S. cerevisiae his4A, his4B, his4C, and his4ABC mutant strains, which indicates that the clone contains the entire HIS4 gene. The gene was isolated on plasmid pSTC7, whose physical map was constructed with BamHI, SalI, and EcoRV restriction endonucleases, locating the HIS4 gene on a 14-kilobase-pair DNA fragment. Hybridization experiments with HIS4 and C. albicans genomic DNA showed correspondence between the restriction patterns of the gene with that of the chromosomal DNA, indicating that the gene originates from C. albicans and appears in a single copy. Chromosomes of C. albicans CBS562 and four other strains were resolved by orthogonal-field alteration gel electrophoresis. The electrokaryotyping results showed heterogeneity in chromosomal sizes. The electrokaryotyping of CBS 562 showed a resolution of six chromosomal bands, three of which seemed to be doublets. The C. albicans HIS4 gene was located on the largest resolvable chromosome in all of the strains.

Purification and in vitro complementation of mutant histidinol dehydrogenases.

The biochemistry of interallelic complementation within the Salmonella typhimurium hisD gene was investigated by in vitro protein complementation of mutant histidinol dehydrogenases (EC 1.1.1.23). Double-mutant strains were constructed containing the hisO1242 (constitutive overproducer) attenuator mutation and selected hisDa or hisDb mutations. Extracts from such hisDa986 and hisDb1799 mutant cells failed to show histidinol dehydrogenase activity but complemented to produce active enzyme. Inactive mutant histidinol dehydrogenases were purified from each of the two mutants by ion-exchange chromatography. Complementation by the purified mutant proteins required the presence of 2-mercaptoethanol and MnCl2, and protein-protein titrations indicated that heterodimers were strongly preferred in mixtures of the complementary mutant enzymes. Neither mutant protein showed negative complementation with wild-type enzyme. The Vmax for hybrid histidinol dehydrogenase was 11% of that for native enzyme, with only minor changes in Km values for substrate or coenzyme. Both purified mutant proteins failed to catalyze NAD-NADH exchange reactions reflective of the first catalytic step of the two-step reaction. The inactive enzymes bound 54Mn2+ weakly or not at all in the presence of 2-mercaptoethanol, in contrast to wild-type enzyme which bound 54Mn2+ to 0.6 sites per monomer under the same conditions. The mutant proteins, like wild-type histidinol dehydrogenase, behaved as dimers on analytical gel filtration chromatography, but dissociated to form monomers in the presence of 2-mercaptoethanol. This effect of 2-mercaptoethanol was prevented by low levels of MnCl2. It thus appears that mutant histidinol dehydrogenase molecules bind metal ion poorly. The complementation procedure may allow for formation of a functional Mn2+-binding site, perhaps at the subunit interface.

Detection and properties of l-histidinol dehydrogenase in wheat germ

The presence and partial characterization of the properties of l-histidinol dehydrogenase (EC 1.1.1.23), the enzyme catalysing the last step in the pathway of histidine biosynthesis, has been described in higher plants for the first time. The activity has been found in cell-free extracts from wheat germ, turnip root, radish root and squash fruit. The enzyme has been partially purified and characterized from extracts of acetone powders of wheat germ. DEAE-cellulose chromatography revealed two peaks of histidinol dehydrogenase activity. In one there was a rapid reduction of NAD + in the absence of histidinol; however, the rate was stimulated by the addition of histidinol. The rate in the absence of substrate became quite low after several min and the histidinol-dependent rate was then easily observed. The second peak of activity did not reduce NAD + unless l-histidinol was present in the assay mixture. The K m s for l-histidinol and NAD + were determined for this latter enzyme. The values obtained at saturating concentrations of the other substrate were l-histidinol, 8.8 μM and NAD +, 0.14 mM. The product of the dehydrogenase reaction was histidine as determined by paper chromatography.

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.

Evolutionary mechanism of adaptation of Arthrobacter histodinolovorans and Pseudomonas aeruginosa to use L-histidinol as a sole source of nitrogen and carbon.

SUMMARY: Mechanisms whereby Arthrobacter histidinolovorans and Pseudomonas aeruginosa grow on L-histidinol as sole source of carbon and nitrogen have been examined. Polyacrylamide gel electrophoresis (PAGE) showed two distinct histidinol dehydrogenase (HDH) bands in partially purified Arthrobacter histidinolovorans extracts: HDH(B) was always present but increased up to twofold by growth with histidine; HDH(I), additionally present only in histidinol-grown organisms, which had up to 10 times the total and specific HDH activity of organisms grown on salts+glucose medium. HDH biosynthesis was not repressed by histidine, glucose+ammonia or glutamate. HDH(I) could have evolved from HDH(B) by gene duplication and modification, with the resulting gene acquiring a new, inducible control mechanism. A mutant, hnc-I, of Pseudomonas aeruginosa grew on histidinol as sole source of carbon and nitrogen, which the wild-type did only poorly. Only one HDH band was found on PAGE of partially purified extracts of either mutant or wild-type, however grown, though histidinol-grown hnc-I showed a more intense band and 60 times the HDH activity of glucose+salts-grown organisms. Histidine, glucose+ammonia or glutamate abolished this HDH increase. Here the adaptation seemed to consist of an additional control mechanism giving rise to high levels of the normal HDH when histidinol was sole source of carbon and nitrogen.