Responses of transcriptome and metabolome in the roots of Pugionium cornutum (L.) Gaertn to exogenously applied phthalic acid.

BACKGROUND: The yield and quality of Pugionium cornutum (L.) Gaertn., a healthy, green vegetable with low sugar and high protein contents and high medicinal value, is severely affected by autotoxicity, which is a leading factor in the formation of plant disease. To help characterize the autotoxicity mechanism of P. cornutum (L.) Gaertn., we performed transcriptomic and metabolic analysis of the roots of P. cornutum (L.) Gaertn. response to phthalic acid, an autotoxin from P. cornutum (L.) Gaertn. RESULTS: In this study, high-throughput sequencing of nine RNA-seq libraries generated from the roots.of P. cornutum (L.) Gaertn. under different phthalic acid treatments yielded 37,737 unigenes. In total, 1085 (703 upregulated and 382 downregulated) and 5998 (4385 upregulated and 1613 downregulated) DEGs were identified under 0.1 and 10 mmol·L- 1 phthalic acid treatment, respectively, compared with the control treatment. Glutathione metabolism was among the top five important enriched pathways. In total, 457 and 435 differentially accumulated metabolites were detected under 0.1 and 10 mmol·L- 1 phthalic acid treatment compared with the control, respectively, of which 223 and 253, respectively, increased in abundance. With the increase in phthalic acid concentration, the accumulation of ten metabolites increased significantly, while that of four metabolites decreased significantly, and phthalic acid, dambonitol, 4-hydroxy-butyric acid, homocitrulline, and ethyl ß-D-glucopyranoside were 100 times more abundant under the 10 mmol·L- 1 phthalic acid treatment than under the control. Seventeen differentially expressed genes significantly associated with phthalic acid content were identified. In addition, the L-histidinol content was highest under 0.1 mmol·L- 1 phthalic acid, and a total of eleven differentially expressed genes were significantly positively correlated with the L-histidinol content, all of which were annotated to heat shock proteins, aquaporins and cysteine proteases. CONCLUSIONS: Accumulation of autotoxins altered the metabolic balance in P. cornutum (L.) Gaertn. and influenced water absorption and carbon and nitrogen metabolism. These important results provide insights into the formation mechanisms of autotoxicity and for the subsequent development of new control measures to improve the production and quality of replanted plants.

Structures of Medicago truncatula L-Histidinol Dehydrogenase Show Rearrangements Required for NAD+ Binding and the Cofactor Positioned to Accept a Hydride.

Plants, lower eukaryotes, bacteria, and archaebacteria synthesise L-histidine (His) in a similar, multistep pathway that is absent in mammals. This makes the His biosynthetic route a promising target for herbicides, antifungal agents, and antibiotics. The last enzyme of the pathway, bifunctional L-histidinol dehydrogenase (HDH, EC 1.1.1.23), catalyses two oxidation reactions: from L-histidinol (HOL) to L-histidinaldehyde and from L-histidinaldehyde to His. Over the course of the reaction, HDH utilises two molecules of NAD+ as the hydride acceptor. The object of this study was the HDH enzyme from the model legume plant, Medicago truncatula (MtHDH). Three crystal structures complexed with imidazole, HOL, and His with NAD+ provided in-depth insights into the enzyme architecture, its active site, and the cofactor binding mode. The overall structure of MtHDH is similar to the two bacterial orthologues whose three-dimensional structures have been determined. The three snapshots, with the MtHDH enzyme captured in different states, visualise structural rearrangements that allow for NAD+ binding for the first time. Furthermore, the MtHDH complex with His and NAD+ displays the cofactor molecule situated in a way that would allow for a hydride transfer.

Sample preparation, crystallization and structure solution of HisC from Mycobacterium tuberculosis.

Histidinolphosphate aminotransferase (HisC; Rv1600) from Mycobacterium tuberculosis was overexpressed in M. smegmatis and purified to homogeneity using nickel-nitrilotriacetic acid metal-affinity and gel-filtration chromatography. Diffraction-quality crystals suitable for X-ray analysis were grown by the hanging-drop vapour-diffusion technique using 30% polyethylene glycol monomethyl ether 2000 as the precipitant. The crystals belonged to the hexagonal space group P3221, with an unusual high solvent content of 74.5%. X-ray diffraction data were recorded to 3.08 Å resolution from a single crystal using in-house Cu Kα radiation. The structure of HisC was solved by the molecular-replacement method using its Corynebacterium glutamicum counterpart as a search model. HisC is a dimer in the crystal as well as in solution.

The missing link in plant histidine biosynthesis: Arabidopsis myoinositol monophosphatase-like2 encodes a functional histidinol-phosphate phosphatase.

Histidine (His) plays a critical role in plant growth and development, both as one of the standard amino acids in proteins, and as a metal-binding ligand. While genes encoding seven of the eight enzymes in the pathway of His biosynthesis have been characterized from a number of plant species, the identity of the enzyme catalyzing the dephosphorylation of histidinol-phosphate to histidinol has remained elusive. Recently, members of a novel family of histidinol-phosphate phosphatase proteins, displaying significant sequence similarity to known myoinositol monophosphatases (IMPs) have been identified from several Actinobacteria. Here we demonstrate that a member of the IMP family from Arabidopsis (Arabidopsis thaliana), myoinositol monophosphatase-like2 (IMPL2; encoded by At4g39120), has histidinol-phosphate phosphatase activity. Heterologous expression of IMPL2, but not the related IMPL1 protein, was sufficient to rescue the His auxotrophy of a Streptomyces coelicolor hisN mutant. Homozygous null impl2 Arabidopsis mutants displayed embryonic lethality, which could be rescued by supplying plants heterozygous for null impl2 alleles with His. In common with the previously characterized HISN genes from Arabidopsis, IMPL2 was expressed in all plant tissues and throughout development, and an IMPL2:green fluorescent protein fusion protein was targeted to the plastid, where His biosynthesis occurs in plants. Our data demonstrate that IMPL2 is the HISN7 gene product, and suggest a lack of genetic redundancy at this metabolic step in Arabidopsis, which is characteristic of the His biosynthetic pathway.

Expression, purification and preliminary X-ray characterization of histidinol phosphate phosphatase.

Histidinol phosphate phosphatase (HisPPase) catalyzes the eighth step of histidine biosynthesis, in which L-histidinol phosphate undergoes dephosphorylation to give histidinol. A recombinant form of the histidinol phosphate phosphatase from Thermus thermophilus HB8 has been expressed in Escherichia coli, purified and crystallized in two crystal forms by the hanging-drop vapour-diffusion technique. Crystal form I belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 84.8, b = 97.2, c = 74.9 A, and crystal form II belongs to the orthorhombic space group C222(1), with unit-cell parameters a = 76.9, b = 157.6, c = 116.7 A. The crystals probably contain two monomers in the asymmetric unit, with V(M) values of 2.57 A(3) Da(-1) for form I and 2.96 A(3) Da(-1) for form II. X-ray data have been collected to 1.70 and 1.75 A resolution for crystal forms I and II, respectively.

Crystal structure of histidinol phosphate aminotransferase (HisC) from Escherichia coli, and its covalent complex with pyridoxal-5'-phosphate and l-histidinol phosphate.

The biosynthesis of histidine is a central metabolic process in organisms ranging from bacteria to yeast and plants. The seventh step in the synthesis of histidine within eubacteria is carried out by a pyridoxal-5'-phosphate (PLP)-dependent l-histidinol phosphate aminotransferase (HisC, EC 2.6.1.9). Here, we report the crystal structure of l-histidinol phosphate aminotransferase from Escherichia coli, as a complex with pyridoxamine-5'-phosphate (PMP) at 1.5 A resolution, as the internal aldimine with PLP, and in a covalent, tetrahedral complex consisting of PLP and l-histidinol phosphate attached to Lys214, both at 2.2 A resolution. This covalent complex resembles, in structural terms, the gem-diamine intermediate that is formed transiently during conversion of the internal to external aldimine.HisC is a dimeric enzyme with a mass of approximately 80 kDa. Like most PLP-dependent enzymes, each HisC monomer consists of two domains, a larger PLP-binding domain having an alpha/beta/alpha topology, and a smaller domain. An N-terminal arm contributes to the dimerization of the two monomers. The PLP-binding domain of HisC shows weak sequence similarity, but significant structural similarity with the PLP-binding domains of a number of PLP-dependent enzymes. Residues that interact with the PLP cofactor, including Tyr55, Asn157, Asp184, Tyr187, Ser213, Lys214 and Arg222, are conserved in the family of aspartate, tyrosine and histidinol phosphate aminotransferases. The imidazole ring of l-histidinol phosphate is bound, in part, through a hydrogen bond with Tyr110, a residue that is substituted by Phe in the broad substrate specific HisC enzymes from Zymomonas mobilis and Bacillus subtilis. Comparison of the structures of the HisC internal aldimine, the PMP complex and the HisC l-histidinol phosphate complex reveal minimal changes in protein or ligand structure. Proton transfer, required for conversion of the gem-diamine to the external aldimine, does not appear to be limited by the distance between substrate and lysine amino groups. We propose that the tetrahedral complex has resulted from non-productive binding of l-histidinol phosphate soaked into the HisC crystals, resulting in its inability to be converted to the external aldimine at the HisC active site.

Studies on the possibility of histidine biosynthesis from histodinol, imidazolepyruvic acid, imidazoleacetica acid, and imidazolelactic acid by mixed ruminal bacteria, protozoa, and their mixture in vitro.

The possibility of histidine (His) synthesis using a main biosynthetic pathway involving histidinol (HDL) and also the recycling capability of imidazolic compounds such as imidazolepyruvic acid (ImPA), imidazoleacetic acid (ImAA), and imidazolelactic acid (ImLA) to produce His were investigated using mixed ruminal bacteria (B), protozoa (P), and a mixture of both (BP) in an in vitro system. Rumen microorganisms were anaerobically incubated at 39 degrees C for 18 h with or without each substrate (2 mM) mentioned. His and other related compounds produced in both the supernatants and hydrolyzates of the incubation were analyzed by high-performance liquid chromatography. B, P, and BP suspensions failed to show His synthesizing ability when incubated with HDL. His was synthesized from ImPA by B, P, and BP. Expressed in units "per gram of microbial nitrogen (MN)", ImPA disappearance was greatest in B (72.7 micromol/g MN per hour), followed by BP (33.13 micromol/g MN per hour) and then P (18.6 micromol/g MN per hour) for the 18-h incubation period. The production of His from ImPA in B (240.0, 275.9, and 261.2 micromol/g MN in 6, 12, and 18 h incubation, respectively) was about 3.5 times higher than that in P (67.3, 83.8, and 72.7 micromol/g MN in 6, 12, and 18 h incubation, respectively). Other metabolites produced from ImPA were ImLA, ImAA, histamine (HTM), and urocanic acid (URA), found in all microbial suspensions. ImLA as a substrate remained without diminution in all microbial suspensions. Although ImAA was found to be degraded to a small extent (3.4-6.3%) only after 18 h incubation, neither His nor other metabolites were detected on the chromatograms. These results have been demonstrated for the first time in rumen microorganisms and suggest that His may be an essential amino acid for rumen microorganisms.

Computational modeling of a binding conformation of the intermediate L-histidinal to histidinol dehydrogenase.

Histidinol dehydrogenase (HDH) is one of the enzymes involved in the L-histidine biosynthesis pathway. HDH is a dimer that contains one Zn2+ ion in each identical subunit. In this study, we predicted a possible binding conformation of the intermediate L-histidinal, which is experimentally not known, using a computational modeling method and three potent HDH inhibitors whose structures are similar to that of L-histidinal. At first, a set of the most probable active conformations of the potent inhibitors was determined using two different pharmacophore mapping techniques, the active analogue approach and the distance comparison method. From the most probable active conformations of the three potent inhibitors, the common parts of the L-histidinal structure were extracted and refined by energy minimization to obtain the binding conformation of L-histidinal. This predicted conformation of L-histidinal agrees with an experimentally determined conformation of L-histidine in a single crystal, suggesting that it is an experimentally acceptable conformation. The capability in this conformation to coordinate a Zn2+ ion was examined by comparing the spatial relative geometry of its functional groups with those of ligands that coordinate with a Zn2+ ion in Zn proteins of the Protein Data Bank. This comparison supported our predicted conformation.

Mechanism of Salmonella typhimurium histidinol dehydrogenase: kinetic isotope effects and pH profiles.

L-Histidinol dehydrogenase catalyzes the biosynthetic oxidation of L-histidinol to L-histidine with sequential reduction of two molecules of NAD. Previous isotope exchange results had suggested that the oxidation of histidinol to the intermediate histidinaldehyde occurred 2-3-fold more rapidly than overall catalysis. In this work, we present kinetic isotope effects (KIE) studies at pH 9.0 and at pH 6.7 with stereospecifically mono- and dideuterated histidinols. The data at pH 9.0 support minimal participation of the first hydride transfer and substantial participation of the second hydride transfer in the overall rate limitation. Stopped-flow experiments with protiated histidinol revealed a small burst of NADH production with stoichiometry of 0.12 per subunit, and 0.25 per subunit with dideuterated histidinol, indicating that the overall first half-reaction was not significantly faster than the second reaction sequence. Results from kcat and kcat/KM titrations with histidinol, NAD, and the alternative substrate imidazolyl propanediol demonstrated an essential base with pKa values between 7.7 and 8.4. In KIE experiments performed at pH 6.7 or with a coenzyme analogue at pH 9. 0, the first hydride transfer became more rate limiting. Kinetic simulations based on rate constants estimated from this work fit well with a mechanism that includes a relatively fast, and thermodynamically unfavorable, hydride transfer from histidinol and a slower, irreversible second hydride transfer from a histidinaldehyde derivative. Thus, although the chemistry of the first hydride transfer is fast, both partial reactions participate in the overall rate limitation.

Mutagenesis of histidinol dehydrogenase reveals roles for conserved histidine residues.

The dimeric zinc metalloenzyme L-histidinol dehydrogenase (HDH) catalyzes an unusual four-electron oxidation of the amino alcohol histidinol via the histidinaldehyde intermediate to the acid product histidine with the reduction of two molecules of NAD. An essential base, with pKa about 8, is involved in catalysis. Here we report site-directed mutagenesis studies to replace each of the five histidine residues (His-98, His-261, His-326, His-366, and His-418) in Salmonella typhimurium with either asparagine or glutamine. In all cases, the overexpressed enzymes were readily purified and behaved as dimers. Substitution of His-261 and His-326 by asparagine caused about 7000- and 500-fold decreases in kcat, respectively, with little change in KM values. Similar loss of activity was also reported for a H261N mutant Brassica HDH [Nagai, A., and Ohta, D. (1994) J. Biochem. 115, 22-25]. Kinetic isotope effects, pH profiles, substrate rescue, and stopped-flow experiments suggested that His-261 and His-326 are involved in proton transfers during catalysis. Sensitivity to metal ion chelator and decreased affinities for metal ions with substitutions at His-261 and His-418 suggested that these two residues are candidates for zinc ion ligands.

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.

Amino acid substitutions in membrane-spanning domains of Hol1, a member of the major facilitator superfamily of transporters, confer nonselective cation uptake in Saccharomyces cerevisiae.

Selection for the ability of Saccharomyces cerevisiae cells to take up histidinol, the biosynthetic precursor to histidine, results in dominant mutations at HOL1. The DNA sequence of HOL1 was determined, and it predicts a 65-kDa protein related to the major facilitator family (drug resistance subfamily) of putative transport proteins. Two classes of mutations were obtained: (i) those that altered the coding region of HOL1, conferring the ability to take up histidinol; and (ii) cis-acting mutations (selected in a mutant HOL1-1 background) that increased expression of the Hol1 protein. The ability to transport histidinol and other cations was conferred by single amino acid substitutions at any of three sites located within putative membrane-spanning domains of the transporter. These mutations resulted in the conversion of a small hydrophobic amino acid codon to a phenylalanine codon. Selection for spontaneous mutations that increase histidinol uptake by such HOL1 mutants resulted in mutations that abolish the putative start codon of a six-codon open reading frame located approximately 171 nucleotides downstream of the transcription initiation site and 213 nucleotides upstream of the coding region of HOL1. This single small upstream open reading frame (uORF) confers translational repression upon HOL1; genetic disruption of the putative start codon of the uORF results in a 5- to 10-fold increase in steady-state amounts of Hol1 protein without significantly affecting the level of HOL1 mRNA expression.

Steady-state kinetics of cabbage histidinol dehydrogenase.

Cabbage histidinol dehydrogenase (HDH) oxidizes L-histidinol to L-histidine through two sequential NAD(+)-linked reactions via an alkaline-labile, L-histidinaldehyde intermediate. The kinetic mechanism of the overall reaction as well as the partial reactions involved in the overall catalysis were investigated at pH 7.2 using L-histidinaldehyde as a substrate. Product inhibition patterns conformed to a Bi Uni Uni Bi Ping Pong mechanism as reported for the HDH from Salmonella typhimurium. Thus, the reaction scheme is ordered with the binding of histidinol first and NAD+ second, and histidine is the last product to be released. The intermediate, L-histidinaldehyde, could be a substrate for both the oxidation and the reduction reactions to produce histidine and histidinol, respectively. L-Histidine was not enzymatically reduced in the presence of NADH, indicating that the reaction to oxidize histidinaldehyde is apparently irreversible. L-Histidinaldehyde exhibited a three times greater binding rate constant than histidinol with a considerably small dissociation constant. These results were in agreement with the observation that histidinaldehyde was not released during the overall reaction. The rate of the reduction of histidinaldehyde to histidinol was almost same as that of the overall oxidation reaction. The overall oxidation from histidinol to histidine proceeded about three times slower than the partial oxidation from histidinaldehyde to histidine, suggesting that the first-half forward reaction is the rate-determining step in the total reaction of cabbage HDH.

Probing the membrane topology of Candida tropicalis cytochrome P450.

The membrane topology of two alkane-inducible cytochromes P450 from the yeast Candida tropicalis, alk1 and alk2, was tested by construction of fusion proteins with part of invertase and histidinol dehydrogenase (invHIS4C) and expression in a Saccharomyces cerevisiae his4 mutant. Depending on the localization of invHIS4C on the endoplasmic reticulum (ER) cytoplasmic or luminal side, the enzyme converts histidinol to histidine and allows the his4 yeast strain to grow on histidinol-supplemented medium. The N-terminal segments of alk1 and alk2 were fused to invHIS4C at three different locations that follow the first alk1 and alk2 transmembrane domains or a second putative transmembrane domain of alk1. The combination of this in vivo assay with subcellular immunoprecipitations of the expressed fusion proteins allowed us to establish that both P450s contain only one transmembrane domain with their N-terminus located in the ER lumen. Deletions performed in these fusion proteins removing the first transmembrane domain of alk1 (delta TM) resulted in a less efficient targeting to the ER membrane but did not prevent their insertion in these membranes. Furthermore deletion of a negatively charged peptide preceding the first alk1 transmembrane domain (delta L) in an invHIS4C protein fused after this domain caused the N-terminal to have a positive net charge and to be oriented in the cytoplasm thus translocating the remaining protein into the ER lumen. The presence of the second hydrophobic segment, however, prevented the complete translocation of this fusion protein into the ER lumen. This study describes the first assessment of P450 membrane topology using an in vivo technique.

Gene inactivation in Lactococcus lactis: histidine biosynthesis.

Lactococcus lactis strains from dairy and nondairy sources were tested for the ability to grow in the absence of histidine. Among 60 dairy strains tested, 56 required histidine, whereas only 1 of 11 nondairy strains had this requirement. Moreover, 10 of the 56 auxotrophic strains were able to grow in the presence of histidinol (Hol+), the immediate histidine precursor. This indicates that adaptation to milk often results in histidine auxotrophy. The histidine operon was detected by Southern hybridization in eight dairy auxotrophic strains tested. A large part of the histidine operon (8 kb, containing seven histidine biosynthetic genes and three unrelated open reading frames [ORFs]) was cloned from an auxotroph, which had an inactive hisD gene, as judged by its inability to grow on histidinol. Complementation analysis of three genes, hisA, hisB, and hisG, in Escherichia coli showed that they also were inactive. Sequence analysis of the cloned histidine region, which revealed 98.6% overall homology with that of the previously analyzed prototrophic strain, showed the presence of frameshift mutations in three his genes, hisC, hisG, and hisH, and two genes unrelated to histidine biosynthesis, ORF3 and ORF6. In addition, several mutations were detected in the promoter region of the operon. Northern (RNA) hybridization analysis showed a much lower amount of the his transcript in the auxotrophic strain than in the prototrophic strain. The mutations detected account for the histidine auxotrophy of the analyzed strain. Certain other dairy auxotrophic strains carry a lower number of mutations, since they were able to revert either to a Hol+ phenotype or to histidine prototrophy.

Inhibition of protein synthesis and heat protection: histidinol-resistant mutant cell lines.

The mechanism of histidinol (HST)-induced heat protection was investigated to test the hypothesis that the cessation of protein synthesis itself is one of the events involved in heat protection. For this study, we isolated three HST-resistant mutant strains. HST (5 mM), which inhibited protein synthesis by 88% in the wild type, caused only 0, 9, and 25% inhibition in three mutants, respectively. The drug, which afforded heat protection, (i.e., a 125-fold increase in survival from 4 x 10(-3) to 5 x 10(-1) after 2 hr at 43 degrees C in wild type), did not protect mutant cells from heat killing. In contrast, cycloheximide (10 micrograms/ml) which inhibited protein synthesis by 95% in both wild type and mutant cell types, protected both cell types from heat killing. Therefore, these results suggest that the cessation of protein synthesis, per se, preventing synthesis of nascent polypeptides, is a major event leading to heat protection.

Increased therapeutic index of antineoplastic drugs in combination with intracellular histamine antagonists.

L-Histidinol, a protein synthesis inhibitor and structural analogue of L-histidine, has been demonstrated in chemotherapy-treated mice to be cytoprotective to normal stem cells but to enhance cytotoxicity to tumor cells. N,N-Diethyl-2-[4-(phenylmethyl) phenoxy]ethanamine.HCl (DPPE) is an antagonist of recently described microsomal and nuclear intracellular histamine receptors implicated in the mediation of proliferation and modulation of prostaglandin synthesis. DPPE is cytotoxic to tumor cells in vitro and cytoprotective to the gut in vivo. Noting the similar pharmacologic profiles for histidinol and DPPE and the structural resemblance between histidinol and histamine, we tested 1) whether binding to intracellular histamine receptors may be important to the action of histidinol, 2) whether there exists a differential effect of DPPE and histidinol on proliferating normal and transformed or malignant cells, and 3) whether DPPE, like histidinol, protects host cells from the effects of chemotherapy while augmenting tumor cell kill in vivo. It was observed that histidinol does compete at intracellular histamine receptors in isolated microsomes and nuclei, but with significantly lower affinity than DPPE. Nevertheless, for each agent, potency at intracellular histamine receptors correlates with potency to inhibit DNA and protein synthesis, without cytotoxicity, in normal mitogen-stimulated murine lymphocytes and to kill transformed mouse lymphocytes or MCF-7 human breast cancer cells. As demonstrated previously for histidinol (1-2 g/kg), DPPE (4 mg/kg) protected murine bone marrow progenitors from doxorubicin or fluorouracil, while doses of 4-50 mg/kg significantly enhanced the antitumor activity of doxorubicin and daunorubicin in murine models of early cancer. One postulate to explain the effects of intracellular histamine receptor ligands is that intracellular histamine mediates DNA and protein synthesis, possibly through a downward modulation of growth-inhibitory prostaglandin levels. Antagonism of the intracellular action of histamine at intracellular histamine receptors by DPPE or histidinol may result in differential perturbations of growth/eicosanoid metabolism in normal and malignant cells, thus forming the basis of a new approach to chemotherapy.

HOL1 mutations confer novel ion transport in Saccharomyces cerevisiae.

Saccharomyces cerevisiae histidine auxotrophs are unable to use L-histidinol as a source of histidine even when they have a functional histidinol dehydrogenase. Mutations in the hol1 gene permit growth of His- cells on histidinol by enhancing the ability of cells to take up histidinol from the medium. Second-site mutations linked to HOL1-1 further increase histidinol uptake. HOL1 double mutants and, to a lesser extent, HOL1-1 single mutants show hypersensitivity to specific cations added to the growth medium, including Na+, Li+, Cs+, Be2+, guanidinium ion, and histidinol, but not K+, Rb+, Ca2+, or Mg2+. The Na(+)-hypersensitive phenotype is correlated with increased uptake and accumulation of this ion. The HOL1-1-101 gene was cloned and used to generate a viable haploid strain containing a hol1 deletion mutation (hol1 delta). The uptake of cations, the dominance of the mutant alleles, and the relative inability of hol1 delta cells to take up histidinol or Na+ suggest that hol1 encodes an ion transporter. The novel pattern of ion transport conferred by HOL1-1 and HOL1-1-101 mutants may be explained by reduced selectivity for the permeant ions.

Salmonella typhimurium histidinol dehydrogenase: complete reaction stereochemistry and active site mapping.

The stereochemistry of the L-histidinol dehydrogenase reaction was determined to be R at NAD for both steps, confirming previous results with a fungal extract [Davies, D., Teixeira, A., & Kenworthy, P. (1972) Biochem. J. 127, 335-343]. NMR analysis of monodeuteriohistidinols produced by histidinol/NADH exchange reactions arising via reversal of the alcohol oxidation reaction indicated a single stereochemistry at histidinol for that step. Comparison of vicinal coupling values of the exchange products with those of L-alaninol and a series of (S)-2-amino-1-alcohols allowed identification of the absolute stereochemistry of monodeuteriohistidinols and showed that histidinol dehydrogenase removes first the pro-S then the pro-R hydrogens of substrate histidinol. The enzyme stereochemistry was confirmed by isotope effects for monodeuteriohistidinols as substrates for the pro-R-specific dehydrogenation catalyzed by liver alcohol dehydrogenase. Active site mapping was undertaken to investigate substrate-protein interactions elsewhere in the histidinol binding site. Critical binding regions are the side-chain amino group and the imidazole ring, whose methylation at the 1- or 2-position caused severe decreases in binding affinity. Use of alternative substrates further clarified active site interactions with the substrate. Compounds in which the alpha-amino group was replaced by chloro, bromo, or hydrogen substituents were not substrates of the overall reaction at 1/10,000 the normal rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Orientation-dependent function of a short CYC1 DNA fragment in directing mRNA 3' end formation in yeast.

We have cloned an 82-base-pair region spanning the site of normal 3' end formation of Saccharomyces cerevisiae CYC1 mRNA into an integrative vector carrying the 5' end of the actin gene (including its intron) fused in frame to HIS4ABC sequences. This vector can confer the ability to grow on histidinol if HIS4C (encoding histidinol dehydrogenase) is sufficiently expressed. With the CYC1 fragment cloned in its wild-type (forward) orientation within the actin intron, transformants cannot grow on histidinol, whereas cells transformed with the vector carrying the reverse orientation of this fragment are able to grow well. RNA transfers demonstrate that transformants containing the forward orientation accumulate less than 40% of the control level of full-length mRNA and reveal the presence of a short, stable (approximately equal to 300 nucleotides) poly(A) RNA that represents 60-70% of the transcripts originating from the same promoter. The reverse orientation of the insert allows near-normal levels of full-length mRNA. Mapping of the 3' end of the truncated RNA indicates that poly(A) addition is variable in length but occurs at the same location as in the normal CYC1 transcript. Dominant and recessive suppressor mutations permit growth on histidinol despite the inserted fragment. Genetic analyses indicate that most of the dominant mutants are cis-acting and that the recessive mutants define a minimum of three complementation groups, indicating that defects in several different genes can restore higher levels of HIS4C expression.