Histidine Degradation Pathway Research Articles

Influence of Histidine Administration on Ammonia and Amino Acid Metabolism: A Review.

Histidine (HIS) is an essential amino acid investigated for therapy of various diseases, used for tissue protection in transplantation and cardiac surgery, and as a supplement to increase muscle performance. The data presented in the review show that HIS administration may increase ammonia and affect the level of several amino acids. The most common are increased levels of alanine, glutamine, and glutamate and decreased levels of glycine and branched-chain amino acids (BCAA, valine, leucine, and isoleucine). The suggested pathogenic mechanisms include increased flux of HIS through HIS degradation pathway (increases in ammonia and glutamate), increased ammonia detoxification to glutamine and exchange of the BCAA with glutamine via L-transporter system in muscles (increase in glutamine and decrease in BCAA), and tetrahydrofolate depletion (decrease in glycine). Increased alanine concentration is explained by enhanced synthesis in extrahepatic tissues and impaired transamination in the liver. Increased ammonia and glutamine and decreased BCAA levels in HIS-treated subjects indicate that HIS supplementation is inappropriate in patients with liver injury. The studies investigating the possibilities to elevate carnosine (beta-alanyl-L-histidine) content in muscles show positive effects of beta-alanine and inconsistent effects of HIS supplementation. Several studies demonstrate HIS depletion due to enhanced availability of methionine, glutamine, or beta-alanine.

Global Regulatory Roles of the Histidine-Responsive Transcriptional Repressor HutC in Pseudomonas fluorescens SBW25.

HutC is known as a transcriptional repressor specific for histidine utilization (hut) genes in Gram-negative bacteria, including Pseudomonas fluorescens SBW25. However, its precise mode of protein-DNA interactions hasn't been examined with purified HutC proteins. Here, we performed electrophoretic mobility shift assay (EMSA) and DNase I footprinting using His6-tagged HutC and biotin-labeled probe of the hut promoter (PhutU). Results revealed a complex pattern of HutC oligomerization, and the specific protein-DNA interaction is disrupted by urocanate, a histidine derivative, in a concentration-dependent manner. Next, we searched for putative HutC-binding sites in the SBW25 genome. This led to the identification of 143 candidate targets with a P value less than 10-4 HutC interaction with eight selected candidate sites was subsequently confirmed by EMSA analysis, including the type IV pilus assembly protein PilZ, phospholipase C (PlcC) for phosphatidylcholine hydrolyzation, and key regulators of cellular nitrogen metabolism (NtrBC and GlnE). Finally, an isogenic hutC deletion mutant was subjected to transcriptome sequencing (RNA-seq) analysis and phenotypic characterization. When bacteria were grown on succinate and histidine, hutC deletion caused upregulation of 794 genes and downregulation of 525 genes at a P value of <0.05 with a fold change cutoff of 2.0. The hutC mutant displayed an enhanced spreading motility and pyoverdine production in laboratory media, in addition to the previously reported growth defect on the surfaces of plants. Together, our data indicate that HutC plays global regulatory roles beyond histidine catabolism through low-affinity binding with operator sites located outside the hut locus.IMPORTANCE HutC in Pseudomonas is a representative member of the GntR/HutC family of transcriptional regulators, which possess a N-terminal winged helix-turn-helix (wHTH) DNA-binding domain and a C-terminal substrate-binding domain. HutC is generally known to repress expression of histidine utilization (hut) genes through binding to the PhutU promoter with urocanate (the first intermediate of the histidine degradation pathway) as the direct inducer. Here, we first describe the detailed molecular interactions between HutC and its PhutU target site in a plant growth-promoting bacterium, P. fluorescens SBW25, and further show that HutC possesses specific DNA-binding activities with many targets in the SBW25 genome. Subsequent RNA-seq analysis and phenotypic assays revealed an unexpected global regulatory role of HutC for successful bacterial colonization in planta.

Cytosolic 10-formyltetrahydrofolate dehydrogenase regulates glycine metabolism in mouse liver

ALDH1L1 (10-formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism highly expressed in liver, metabolizes 10-formyltetrahydrofolate to produce tetrahydrofolate (THF). This reaction might have a regulatory function towards reduced folate pools, de novo purine biosynthesis, and the flux of folate-bound methyl groups. To understand the role of the enzyme in cellular metabolism, Aldh1l1−/− mice were generated using an ES cell clone (C57BL/6N background) from KOMP repository. Though Aldh1l1−/− mice were viable and did not have an apparent phenotype, metabolomic analysis indicated that they had metabolic signs of folate deficiency. Specifically, the intermediate of the histidine degradation pathway and a marker of folate deficiency, formiminoglutamate, was increased more than 15-fold in livers of Aldh1l1−/− mice. At the same time, blood folate levels were not changed and the total folate pool in the liver was decreased by only 20%. A two-fold decrease in glycine and a strong drop in glycine conjugates, a likely result of glycine shortage, were also observed in Aldh1l1−/− mice. Our study indicates that in the absence of ALDH1L1 enzyme, 10-formyl-THF cannot be efficiently metabolized in the liver. This leads to the decrease in THF causing reduced generation of glycine from serine and impaired histidine degradation, two pathways strictly dependent on THF.

Impact of Aldh1l1 Knockout On Metabolic Phenotype in Mouse Liver

ALDH1L1 (10‐formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism, is highly expressed in liver. This enzyme metabolizes 10‐formyltetrahydrofolate to produce tetrahydrofolate (THF) and is involved in the regulation of reduced folate pools, de novo purine biosynthesis, and the flux of folate‐bound methyl groups. The goal of this study was to survey the metabolic impact of the Aldh1l1 gene loss in mouse liver. Aldh1l1−/− (KO) mice were generated using gene‐targeted ES cell clone (C57BL/6N background) from KOMP repository. KO mice were viable and did not show obvious phenotypic differences from the wild type animals. Flash frozen hepatic tissues from 12–14 weeks old Aldh1l1+/+and Aldh1l1−/− mice were profiled to identify metabolite changes and define associated pathways. In this study, mice were kept on standard AIN‐93 diet with 2.2 ppm of folic acid. Global metabolomic analysis was performed by Metabolon Inc. (Durham, NC) using Ultrahigh Performance Liquid Chromatography‐Tandem Mass Spectroscopy (UPLC‐MS/MS) for five samples from each group. This analysis has identified 628 known metabolites and principal component analysis demonstrated good segregation between the two genotypes. The difference for 91 metabolites was statistically significant between the two groups, with 31 compounds being decreased and 60 being elevated in Aldh1l1−/− compared to the Aldh1l1+/+ mice. This analysis indicated that KO mice might experience functional folate deficiency. Specifically, the intermediate of the histidine degradation pathway serving as a marker of folate deficiency, formiminoglutamate, was increased more than 15‐fold in KO mice. Furthermore, tissue folic acid and dihydrofolate were strongly depleted (more than 2‐fold and 5‐fold, respectively) in these animals, which would be in agreement with folate deficiency. In addition, a 2‐fold decrease in glycine was detected in KO mice. Strong drop in several glycine conjugates was also observed in the KO genotype, which is apparently associated with the deficiency of glycine. The depletion of glycine was likely caused by the decrease of the THF‐dependent synthesis of glycine from serine, with the underlying basis for this phenomenon being the decrease of the THF pool observed in Aldh1l1−/− mice. The depletion of glycine and/or folate could be the cause of several secondary effects on metabolism including the decrease in glutathione, lower availability of ascorbate, impaired mitochondrial lipid metabolism, oxidative stress, and less efficient remediation of toxic products. Our data also imply a compromised liver function in the KO mice and associated changes in gut microbiome activity. Overall, our study indicated significant metabolic perturbations caused by the Aldh1l1 gene knockout.Support or Funding InformationNIH R01DK 54388This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Histidine catabolism is a major determinant of methotrexate sensitivity.

The chemotherapeutic drug methotrexate inhibits the enzyme DHFR (dihydrofolate reductase)1, which generates tetrahydrofolate (THF), an essential cofactor in nucleotide synthesis2. Depletion of THF causes cell death by suppressing DNA and RNA production3. While methotrexate is widely used as an anti-cancer agent and the subject of over a thousand ongoing clinical trials4, its high toxicity often leads to the premature termination of its use, diminishing its potential efficacy5. To identify genes that modulate the response of cancer cells to methotrexate, we performed a CRISPR/Cas9-based screen6,7. This screen yielded FTCD, which encodes an enzyme (formimidoyltransferase cyclodeaminase) needed for the catabolism of the amino acid histidine8, a process not previously linked to methotrexate sensitivity. In cultured cancer cells, depletion of multiple genes in the histidine catabolism pathway dramatically decreased sensitivity to methotrexate. Mechanistically, histidine catabolism drains the cellular pool of THF, which is particularly detrimental to methotrexate-treated cells. Moreover, expression of the rate-limiting enzyme in histidine catabolism is associated with methotrexate sensitivity in cancer cell lines and with survival rate in patients. In vivo dietary supplementation of histidine increased flux through the histidine degradation pathway and enhanced the sensitivity of leukemia xenografts to methotrexate. Thus, the histidine degradation pathway significantly influences the sensitivity of cancer cells to methotrexate and may be exploited to improve methotrexate efficacy through a simple dietary intervention.

PREDICTING THE BIODEGRADABILITY NATURE OF IMIDAZOLE AND ITS DERIVATIVES BY MODULATING TWO HISTIDINE DEGRADATION ENZYMES (UROCANASE AND FORMIMINOGLUTAMASE) ACTIVITIES

Objectives: The biodegradation pathway of substituted imidazole ring compounds has been reported to have close analogy to the histidine degradation pathway. This prompted the present study to be carried out on 12 selected imidazole and its derivatives which are 1-imidazole, 1, 2-dimethylimidazole, 1-ethyl imidazole, 2-ethyl-4-methylimidazole, 2-isopropylimidazole, 2-Isopropyl-4-nitro-1H-imidazole, 1-methylimidazole, 2-methyl-5-nitroimidazole, 2-methyl-1-vinylimidazole, 1-nitro imidazole, 1-phenyl imidazole, and 1-vinylimidazole.Methods: The imidazole and its derivatives were evaluated on the docking behavior of urocanase and formiminoglutamase using PatchDock. In addition, molecular physicochemical, drug-likeness, absorption, distribution, metabolism, and excretion analyses (ADME) were done.Results: The molecular physicochemical analysis revealed that all the tested ligands showed nil violation and complied well with the Lipinski’s rule of five. ADME analysis showed that 1-phenylimidazole alone predicated to have cytochrome P450 1A2 inhibition effect. Docking studies revealed that 1-nitroimidazole showed the least atomic contact energy with both targeted enzymes (urocanase and FIGase).Conclusion: Inhibition of both enzymes (urocanase and FIGase) might show poor biodegradability nature. Thus, we can predict biodegradability nature of imidazloe and its derivatives by modulating two histidine degradation enzymes activities.

Crystallization and X Ray Diffraction Data Analyses of the Enzyme Urocanate Hydratase fromTrypanosoma cruzi

Trypanosoma cruzi is the only trypanosomatid pathogenic to humans in which the four enzymes of the histidine degradation pathway were found up to now. The enzyme urocanate hydratase from Trypanosoma cruzi catalyzes the second step of histidine degradation and is essential for the catabolic conversion of urocanate to 4-imidazolone-5-propionate, which finally yields glutamate and further glutamine and tricarboxylic acid cycle intermediates such as alpha-ketoglutarate. The recombinant C-terminally His6-tagged protein was expressed in Escherichia coli , purified in a homogenous form and crystallized in several conditions, with the best crystals obtained with 0.04 M dipotassium hydrogen phosphate, 16.00 % ( w / v ) polyethylene glycol 8,000 and 22.00 % ( v / v ) glycerol. X ray diffraction data were collected to 2.61 A resolution. The crystals belong to the monoclinic space group P2 1 , with unit cell parameters a = 85.12, b = 137.63, c = 117.69 A, beta = 94.69 ° and are suitable for molecular replacement phasing. The asymmetric unit contains four monomers, with a V M of 2.3 A 3 /Da and a solvent content of 45.9 %. DOI: 10.5935/1984-6835.20160051

Structure of N-formimino-L-glutamate iminohydrolase from Pseudomonas aeruginosa.

N-Formimino-l-glutamate iminohydrolase (HutF), from Pseudomonas aeruginosa with a locus tag of Pa5106 (gi|15600299), is a member of the amidohydrolase superfamily. This enzyme catalyzes the deamination of N-formimino-l-glutamate to N-formyl-l-glutamate and ammonia in the histidine degradation pathway. The crystal structure of Pa5106 was determined in the presence of the inhibitors N-formimino-l-aspartate and N-guanidino-l-glutaric acid at resolutions of 1.9 and 1.4 Å, respectively. The structure of an individual subunit is composed of two domains with the larger domain folding as a distorted (β/α)8-barrel. The (β/α)8-barrel domain is composed of eight β-strands flanked by 11 α-helices, whereas the smaller domain is made up of eight β-strands. The active site of Pa5106 contains a single zinc atom that is coordinated by His-56, His-58, His-232, and Asp-320. The nucleophilic solvent water molecule coordinates with the zinc atom at a distance of 2.0 Å and is hydrogen bonded to Asp-320 and His-269. The α-carboxylate groups of both inhibitors are hydrogen bonded to the imidazole moiety of His-206, the hydroxyl group of Tyr-121, and the side chain amide group of Gln-61. The side chain carboxylate groups of the two inhibitors are ion-paired with the guanidino groups of Arg-209 and Arg-82. Computational docking of high-energy tetrahedral intermediate forms of the substrate, N-formimino-l-glutamate, to the three-dimensional structure of Pa5106 suggests that this compound likely undergoes a re-faced nucleophilic attack at the formimino group by the metal-bound hydroxide. A catalytic mechanism of the reaction catalyzed by Pa5106 is proposed.

Histidine degradation via an aminotransferase increases the nutritional flexibility of Candida glabrata.

The ability to acquire nutrients during infections is an important attribute in microbial pathogenesis. Amino acids are a valuable source of nitrogen if they can be degraded by the infecting organism. In this work, we analyzed histidine utilization in the fungal pathogen of humans Candida glabrata. Hemiascomycete fungi, like C. glabrata or Saccharomyces cerevisiae, possess no gene coding for a histidine ammonia-lyase, which catalyzes the first step of a major histidine degradation pathway in most other organisms. We show that C. glabrata instead initializes histidine degradation via the aromatic amino acid aminotransferase Aro8. Although ARO8 is also present in S. cerevisiae and is induced by extracellular histidine, the yeast cannot use histidine as its sole nitrogen source, possibly due to growth inhibition by a downstream degradation product. Furthermore, C. glabrata relies only on Aro8 for phenylalanine and tryptophan utilization, since ARO8, but not its homologue ARO9, was transcriptionally activated in the presence of these amino acids. Accordingly, an ARO9 deletion had no effect on growth with aromatic amino acids. In contrast, in S. cerevisiae, ARO9 is strongly induced by tryptophan and is known to support growth on aromatic amino acids. Differences in the genomic structure of the ARO9 gene between C. glabrata and S. cerevisiae indicate a possible disruption in the regulatory upstream region. Thus, we show that, in contrast to S. cerevisiae, C. glabrata has adapted to use histidine as a sole source of nitrogen and that the aromatic amino acid aminotransferase Aro8, but not Aro9, is the enzyme required for this process.

A Common Catalytic Mechanism for Proteins of the HutI Family

Imidazolonepropionase (HutI) (imidazolone-5-propanote hydrolase, EC 3.5.2.7) is a member of the amidohydrolase superfamily and catalyzes the conversion of imidazolone-5-propanoate to N-formimino-L-glutamate in the histidine degradation pathway. We have determined the three-dimensional crystal structures of HutI from Agrobacterium tumefaciens (At-HutI) and an environmental sample from the Sargasso Sea Ocean Going Survey (Es-HutI) bound to the product [ N-formimino-L-glutamate (NIG)] and an inhibitor [3-(2,5-dioxoimidazolidin-4-yl)propionic acid (DIP)], respectively. In both structures, the active site is contained within each monomer, and its organization displays the landmark feature of the amidohydrolase superfamily, showing a metal ligand (iron), four histidines, and one aspartic acid. A catalytic mechanism involving His265 is proposed on the basis of the inhibitor-bound structure. This mechanism is applicable to all HutI forms.

Genetic Analysis of the Histidine Utilization (hut) Genes in Pseudomonas fluorescens SBW25

The histidine utilization (hut) locus of Pseudomonas fluorescens SBW25 confers the ability to utilize histidine as a sole carbon and nitrogen source. Genetic analysis using a combination of site-directed mutagenesis and chromosomally integrated lacZ fusions showed the hut locus to be composed of 13 genes organized in 3 transcriptional units: hutF, hutCD, and 10 genes from hutU to hutG (which includes 2 copies of hutH, 1 of which is nonfunctional). Inactivation of hutF eliminated the ability to grow on histidine, indicating that SBW25 degrades histidine by the five-step enzymatic pathway. The 3 hut operons are negatively regulated by the HutC repressor with urocanate (the first intermediate of the histidine degradation pathway) as the physiological inducer. 5'-RACE analysis of transcriptional start sites revealed involvement of both sigma(54) (for the hutU-G operon) and sigma(70) (for hutF); the involvement of sigma(54) was experimentally demonstrated. CbrB (an enhancer binding protein for sigma(54) recruitment) was required for bacterial growth on histidine, indicating positive control of hut gene expression by CbrB. Recognition that a gene (named hutD) encoding a widely distributed conserved hypothetical protein is transcribed along with hutC led to analysis of its role. Mutational and gene fusion studies showed that HutD functions independently of HutC. Growth and fitness assays in laboratory media and on sugar beet seedlings suggest that HutD acts as a governor that sets an upper bound to the level of hut activity.

A Catalytic Mechanism Revealed by the Crystal Structures of the Imidazolonepropionase from Bacillus subtilis

Imidazolonepropionase (EC 3.5.2.7) catalyzes the third step in the universal histidine degradation pathway, hydrolyzing the carbon-nitrogen bonds in 4-imidazolone-5-propionic acid to yield N-formimino-l-glutamic acid. Here we report the crystal structures of the Bacillus subtilis imidazolonepropionase and its complex at 2.0-A resolution with substrate analog imidazole-4-acetic acid sodium (I4AA). The structure of the native enzyme contains two domains, a TIM (triose-phosphate isomerase) barrel domain with two insertions and a small beta-sandwich domain. The TIM barrel domain is quite similar to the members of the alpha/beta barrel metallo-dependent hydrolase superfamily, especially to Escherichia coli cytosine deaminase. A metal ion was found in the central cavity of the TIM barrel and was tightly coordinated to residues His-80, His-82, His-249, Asp-324, and a water molecule. X-ray fluorescence scan analysis confirmed that the bound metal ion was a zinc ion. An acetate ion, 6 A away from the zinc ion, was also found in the potential active site. In the complex structure with I4AA, a substrate analog, I4AA replaced the acetate ion and contacted with Arg-89, Try-102, Tyr-152, His-185, and Glu-252, further defining and confirming the active site. The detailed structural studies allowed us to propose a zinc-activated nucleophilic attack mechanism for the hydrolysis reaction catalyzed by the enzyme.

Mechanistic Characterization of N-Formimino-l-glutamate Iminohydrolase from Pseudomonas aeruginosa

N-Formimino-l-glutamate iminohydrolase (HutF) from Pseudomonas aeruginosa catalyzes the deimination of N-formimino-l-glutamate in the histidine degradation pathway. An amino acid sequence alignment between HutF and members of the amidohydrolase superfamily containing mononuclear metal centers indicated that residues Glu-235, His-269, and Asp-320 are involved in substrate binding and activation of the nucleophilic water molecule. The purified enzyme contained up to one equivalent of zinc. The metal was removed by dialysis against the metal chelator dipicolinate with the complete loss of catalytic activity. Enzymatic activity was restored by incubation of the apoprotein with Zn2+, Cd2+, Ni2+, or Cu2+. The mutation of Glu-235, His-269, or Asp-320 resulted in the diminution of catalytic activity by two to six orders of magnitude. Bell-shaped profiles were observed for kcat and kcat/Km as a function of pH. The pKa of the group that must be unprotonated for catalytic activity was consistent with the ionization of His-269. This residue is proposed to function as a general base in the abstraction of a proton from the metal-bound water molecule. In the proposed catalytic mechanism, the reaction is initiated by the abstraction of a proton from the metal-bound water molecule by the side chain imidazole of His-269 to generate a tetrahedral intermediate of the substrate. The collapse of the tetrahedral intermediate commences with the abstraction of a second proton via the side chain carboxylate of Asp-320. The C-N bond of the substrate is subsequently cleaved with proton transfer from His-269 to form ammonia and the N-formyl product. The postulated role of the invariant Glu-235 is to ion pair with the positively charged formimino group of the substrate.

Annotating Enzymes of Unknown Function: N-Formimino-l-glutamate Deiminase Is a Member of the Amidohydrolase Superfamily

The functional assignment of enzymes that catalyze unknown chemical transformations is a difficult problem. The protein Pa5106 from Pseudomonas aeruginosa has been identified as a member of the amidohydrolase superfamily by a comprehensive amino acid sequence comparison with structurally authenticated members of this superfamily. The function of Pa5106 has been annotated as a probablechlorohydrolase or cytosine deaminase. A close examination of the genomic content of P. aeruginosa reveals that the gene for this protein is in close proximity to genes included in the histidine degradation pathway. The first three steps for the degradation of histidine include the action of HutH, HutU, and HutI to convert L-histidine to N-formimino-L-glutamate. The degradation of N-formimino-L-glutamate to L-glutamate can occur by three different pathways. Three proteins in P. aeruginosa have been identified that catalyze two of the three possible pathways for the degradation of N-formimino-L-glutamate. The protein Pa5106 was shown to catalyze the deimination of N-formimino-L-glutamate to ammonia and N-formyl-L-glutamate, while Pa5091 catalyzed the hydrolysis of N-formyl-L-glutamate to formate and L-glutamate. The protein Pa3175 is dislocated from the hut operon and was shown to catalyze the hydrolysis of N-formimino-L-glutamate to formamide and L-glutamate. The reason for the coexistence of two alternative pathways for the degradation of N-formimino-L-glutamate in P. aeruginosa is unknown.

Protein preparation, crystallization and preliminary X-ray analysis of imidazolonepropionase from Bacillus subtilis

Imidazolonepropionase (EC 3.5.2.7) is the third enzyme of the histidine degradation pathway that has been conserved from bacteria to eukaryotes. The enzyme is the only one with unknown three-dimensional structure in this pathway. In this work, Bacillus subtilis imidazolonepropionase (HutI) was expressed in E. coli and purified to homogeneity. After thrombin digestion, high quality crystals were obtained by hanging-drop vapor diffusion method. The best crystal diffracted to 2.0 Å and belonged to the space group P2 1 with unit-cell parameters a = 57.73 Å, b = 106.34 Å, c = 66.47 Å, β = 89.93 °.

Amino acid utilization by Chlamydomonas reinhardtii: specific study of histidine

Phytoplankton live in fluctuating environments where many factors such as grazing pressure, sinking, light availability, nutrient uptake and turnover influence the distribution of phytoplankton in time and space. The purpose of this study was to investigate if under conditions of depletion of inorganic nitrogen, as recorded in summer in naturals waters, phytoplanktonic species have the capability of using organic nitrogen sources, including free or combined amino acids, in addition to inorganic nitrogen. The study has focussed on histidine, the degradation of which yielding potentially three nitrogen atoms for each molecule of histidine. Chlamydomonas reinhardtii (CCAP 11/32A) was cultivated axenically with two different sources of nitrogen (histidine and/or ammonium). In the presence of histidine as sole source of nitrogen, cell growth was comparable to that observed with the same concentration of nitrogen in ammonium form. In the presence of both histidine and ammonium, histidine degradation was observed only when the concentration of ammonium was depleted. Under these conditions, the first two enzymes of histidine degradation pathway, histidase (EC 4.3.1.3) and urocanase (EC 4.2.1.49) were produced and were co-ordinately regulated. Histidase activity was also controlled by succinate and glutamate as carbon sources. Histidase was purified 1018-fold and partially characterized. The molecular weight of the native enzyme was estimated to 152.4 kDa corresponding to four subunits of 38.1 kDa. The enzyme did not exhibit classical Michaelis–Menten kinetics but showed a relationship between the rate of catalysis ( V) and the concentration of substrate ( S), characteristic of negative allosteric behavior. A Hill coefficient of 4 was measured for histidine concentrations higher than 20.5 mM.

Crystallization and preliminary X-ray analysis of the formiminotransferase domain from the bifunctional enzyme formiminotransferase-cyclodeaminase.

Formiminotransferase-cyclodeaminase (E.C. 2.1.2.5-E.C. 4.3.1.4) is a bifunctional enzyme involved in the histidine-degradation pathway which exhibits specificity for polyglutamylated folate substrates. The first function of the enzyme transfers the formimino group of formiminoglutamate to the N5 position of tetrahydrofolate, while the second function catalyses the cyclodeamination of the formimino group, yielding N5,10-methenyl-tetrahydrofolate, with efficient channeling of the intermediate between these activities. Initial studies have shown that the enzyme consists of eight identical subunits of 62 kDa each, arranged as a circular tetramer of dimers. It is this formation which results in two different dimeric interfaces, which are necessary for the two different activities. The identical subunits have been shown to consist of two domains, each of which can be obtained as dimers. The formiminotransferase domain has been crystallized in the presence of the substrate analogue folinic acid. The crystals belong to space group P212121, with unit-cell dimensions a = 64.4, b = 103.7, c = 122.3 A. Both a native data set and a mercurial derivative data set have been collected to 2.8 A resolution.

A Formiminotransferase Cyclodeaminase Isoform Is Localized to the Golgi Complex and Can Mediate Interaction of Trans-Golgi Network-derived Vesicles with Microtubules

A protein of 60 kDa (p60) has been identified using a quantitative in vitro vesicle-microtubule binding assay. Purified p60 induces co-sedimentation with microtubules of trans-Golgi network-derived vesicles isolated from polarized, perforated Madin-Darby canine kidney cells. Sequencing of the cDNA coding for this protein revealed that it is the chicken homologue of formiminotransferase cyclodeaminase (FTCD), a liver-specific enzyme involved in the histidine degradation pathway. Purified p60 from chicken liver has formiminotransferase activity, confirming that it is FTCD or an isoform of this enzyme. Isoforms of FTCD were identified in chicken hepatoma and HeLa cells, and immunolocalize to the region of the Golgi complex and vesicular structures in its vicinity. Furthermore, 58K, a previously identified microtubule-binding Golgi protein from rat liver (Bloom, G. S., and Brashear, T. A. (1989) J. Biol. Chem. 264, 16083-16092), is identical to FTCD. Both proteins co-purify with microtubules and co-localize with membranes of the Golgi complex. The capacity of FTCD to bind both to microtubules and Golgi-derived membranes may suggest that this protein, or one of its isoforms, might have in addition to its enzymatic activity, a second physiological function in mediating interaction of Golgi-derived membranes with microtubules.

Histidine Utilization by the Unicellular Alga Dunaliella tertiolecta

The unicellular alga Dunaliella tertiolecta was cultivated axenically with histidine, or ammonium, or histidine and ammonium ions as nitrogen sources. In the presence of histidine as the sole source of nitrogen, cell growth is comparable to that observed with the same concentration of nitrogen in ammonium form. In the presence of both histidine and ammonium, histidine degradation is observed only when the concentration of ammonium falls below 4 mM. Under these conditions, the first two enzymes of histidine degradation pathway, histidase and urocanase, as well as histidine permease, are produced. Linear relationships are observed between histidase and urocanase and histidase and histidine permease, respectively, showing that, in D. tertiolecta enzyme, systems are coordinately regulated. Histidase activity is also controlled by succinate, glutamate and acetate as carbon sources. D. tertiolecta is unable to grow on a medium containing acetate and histidine together in the absence of ammonium.

The degradation of L-histidine, imidazolyl-L-lactate and imidazolylpropionate by Pseudomonas testosteroni.

1. Imidazol-5-ylpropionate and imidazol-5-yl-lactate are degraded by Pseudomonas testosteroni via inducible pathways. 2. Growth on either compound as the sole source of carbon results in the induction of the enzymes for histidine catabolism. 3. The pathway of histidine degradation in this organism, a non-fluorescent Pseudomonad, is shown to be the same as that operating in Pseudomonas fluorescens and Pseudomonas putida. It consists of the successive formation of urocanate, imidazol-4-on-5-ylpropionate, N-formimino-l-glutamate, N-formyl-l-glutamate and glutamate. 4. Whole cells of P. testosteroni accumulate urocanate in the reaction mixture when incubated with imidazolylpropionate, but only after an adaptive lag period which is removed by previous growth on imidazolylpropionate as the source of carbon. 5. Imidazolyl-lactate is oxidized to imidazolylpyruvate, which then gives rise to histidine by specific transamination with l-glutamate. 6. Cells grown on histidine, urocanate or imidazolylpropionate are also able to degrade imidazolyllactate. 7. Mutants lacking urocanase are unable to grow on imidazolylpropionate, imidazolyl-lactate, histidine or urocanate. One with impaired histidase activity cannot utilize histidine or imidazolyl-lactate, but grows normally on imidazolylpropionate or urocanate. A mutant unable to grow on imidazolylpropionate is indistinguishable from the wild-type with respect to growth on histidine, imidazolyl-lactate or urocanate. 8. Thus it is established that imidazolyl-lactate is metabolized via histidine whereas imidazolylpropionate enters the histidine degradation pathway after conversion into urocanate.