The self-assembly of L-histidine might be the cause of histidinemia.

L-Histidine is an essential amino acid with unique biochemical and physiological properties. Histidinemia is a disease condition caused by the elevated level of L-histidine in our blood. Mutations in the histidase, an enzyme for the breakdown of histidine, is the cause of the rise in histidine concentration. To our knowledge, no research has been done on why a high concentration of histidine causes histidinemia. In this study, we provide a potential explanation why the elevated levels of histidine in the human body causes histidinemia. In this study we have found that L-histidine self-assembled in water to form nano sheet structures at physiological pH and temperature, using 1D 1H NMR spectroscopy, diffusion ordered spectroscopy (DOSY) and scanning electron microscope (SEM) techniques. The kinetics of self-assembly has been studied using real time NMR spectroscopy. We observed that both the aromatic ring and aliphatic part are equally contributing to the self-assembly of L-histidine. The symptoms of histidinemia, neurological deficits and speech delays, are similar to that of the neurodegenerative diseases caused by the self-assembly of peptides and proteins. We speculate that the self-assembly of L-histidine might be the cause of histidinemia.

The Histidine Ammonia Lyase of Trypanosoma cruzi Is Involved in Acidocalcisome Alkalinization and Is Essential for Survival under Starvation Conditions.

Trypanosoma cruzi, the agent of Chagas disease, accumulates polyphosphate (polyP) and Ca2+ inside acidocalcisomes. The alkalinization of this organelle stimulates polyP hydrolysis and Ca2+ release. Here, we report that histidine ammonia lyase (HAL), an enzyme that catalyzes histidine deamination with production of ammonia (NH3) and urocanate, is responsible for acidocalcisome alkalinization. Histidine addition to live parasites expressing HAL fused to the pH-sensitive emission biosensor green fluorescent protein (GFP) variant pHluorin induced alkalinization of acidocalcisomes. PolyP decreased HAL activity of epimastigote lysates or the recombinant protein but did not cause its polyphosphorylation, as determined by the lack of HAL electrophoretic shift on NuPAGE gels using both in vitro and in vivo conditions. We demonstrate that HAL binds strongly to polyP and localizes to the acidocalcisomes and cytosol of the parasite. Four lysine residues localized in the HAL C-terminal region are instrumental for its polyP binding, its inhibition by polyP, its function inside acidocalcisomes, and parasite survival under starvation conditions. Expression of HAL in yeast deficient in polyP degradation decreased cell fitness. This effect was enhanced by histidine and decreased when the lysine-rich C-terminal region was deleted. In conclusion, this study highlights a mechanism for stimulation of acidocalcisome alkalinization linked to amino acid metabolism. IMPORTANCE Trypanosoma cruzi is the etiologic agent of Chagas disease and is characterized by the presence of acidocalcisomes, organelles rich in phosphate and calcium. Release of these molecules, which are necessary for growth and cell signaling, is induced by alkalinization, but a physiological mechanism for acidocalcisome alkalinization was unknown. In this work, we demonstrate that a histidine ammonia lyase localizes to acidocalcisomes and is responsible for their alkalinization.

Long non-coding RNA HAL suppresses the migration and invasion of serous ovarian cancer by inhibiting EMT signaling pathway.

OBJECTIVE: To investigate the specific function of long non-coding RNA HAL in serous ovarian cancer (SOC) and to further clarify the regulation of HAL on EMT pathway. MATERIALS AND METHODS: The expression of HAL and TWIST1 was detected by qRT-PCR. CCK8 assay, wound healing assay, transwell assay and flow cytometry were used to detect the HAL function on proliferation, migration, invasion and apoptosis in SOC cells. Western blot was used to calculate protein level of Vimentin, N-cadherin and E-cadherin. The effect of HAL on tumorigenesis of SOC was confirmed by xenograft nude mice model. RESULTS: HAL was significantly decreased in SOC tissues and cells. Overexpression of HAL inhibited the proliferation, migration and invasion of SKOV3 cells, but promoted apoptosis. Furthermore, overexpression of HAL decreased the mRNA and protein levels of TWIST1 via a binding between HAL and TWIST1. Forced expression of TWIST1 reversed the inhibitory role of HAL on SOC cells' migration and invasion. The in vivo tumor growth assay showed that HAL suppressed SOC tumorigenesis with inhibiting EMT pathway. CONCLUSIONS: Our research emphasized HAL acting as a tumor-inhibiting gene by regulating EMT signaling pathway, thus providing some novel experimental basis for clinical treatment of SOC.

Genetic variants in glutamine metabolic pathway genes predict cutaneous melanoma-specific survival.

Glutamine dependence is a unique metabolic defect seen in cutaneous melanoma (CM), directly influencing the treatment and prognosis. Here, we investigated the associations between 6025 common single-nucleotide polymorphisms (SNPs) in 77 glutamine metabolic pathway genes with CM-specific survival (CMSS) using genotyping datasets from two published genome-wide association studies (GWASs). In the single-locus analysis, 76 SNPs were found to be significantly associated with CMSS (P < .050, false-positive report probability < 0.2 and Bayesian false discovery probability < 0.8) in the discovery dataset, of which seven SNPs were replicated in the validation dataset and three SNPs (HAL rs17676826T > C, LGSN rs12663017T > A, and NOXRED1 rs8012548A > G) independently predicted CMSS, with an effect-allele attributed adjusted hazards ratio of 1.52 (95% confidence interval = 1.19-1.93) and P < .001, 0.68 (0.54-0.87) and P = .002 and 0.62 (0.46-0.83) and P = .002, respectively. The model including the number of unfavorable genotypes (NUGs) of these three SNPs and covariates improved the five-year CMSS prediction (P = .012) than the one with other covariates only. Further expression quantitative trait loci (eQTL) analysis found that the LGSN rs12663017 A allele was significantly associated with increased messenger RNA (mRNA) expression levels (P = 8.89 × 10 -11 ) in lymphoblastoid cell lines of the 1000 Genomes Project database. In the analysis of the genotype tissue expression (GTEx) project datasets, HAL rs17676826 C and NOXRED1 rs8012548 G alleles were significantly associated with their mRNA expression levels in sun-exposed skin of the lower leg (P = 6.62 × 10-6 and 1.37 × 10-7 , respectively) and in sun-not-exposed suprapubic skin (P < .001 and 1.43 × 10-8 , respectively). Taken together, these genetic variants of glutamine-metabolic pathway genes may be promising predictors of survival in patients with CM.

Pseudomonas fluorescens Strain R124 Encodes Three Different MIO Enzymes.

A number of class I lyase-like enzymes, including aromatic ammonia-lyases and aromatic 2,3-aminomutases, contain the electrophilic 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) catalytic moiety. This study reveals that Pseudomonas fluorescens R124 strain isolated from a nutrient-limited cave encodes a histidine ammonia-lyase, a tyrosine/phenylalanine/histidine ammonia-lyase (XAL), and a phenylalanine 2,3-aminomutase (PAM), and demonstrates that an organism under nitrogen-limited conditions can develop novel nitrogen fixation and transformation pathways to enrich the possibility of nitrogen metabolism by gaining a PAM through horizontal gene transfer. The novel MIO enzymes are potential biocatalysts in the synthesis of enantiopure unnatural amino acids. The broad substrate acceptance and high thermal stability of PfXAL indicate that this enzyme is highly suitable for biocatalysis.

Bidirectional reporter assay using HAL promoter and TOPFLASH improves specificity in high-throughput screening of Wnt inhibitors.

Constitutive activation of Wnt signaling plays an important role in colorectal and liver tumorigenesis. Cell-based assays using synthetic TCF/LEF (T-cell factor/lymphoid enhancer factor) reporters, as readouts of ß-catenin/TCF-dependent transcriptional activity, have contributed greatly to the discovery of small molecules that modulate Wnt signaling. In the present study, we report a novel screening method, called a bidirectional dual reporter assay. Integrated transcriptome analysis identified a histidine ammonia-lyase gene (HAL) that was negatively regulated by ß-catenin/TCF-dependent transcriptional activity. We leveraged a promoter region of the HAL gene as another transcriptional readout of Wnt signaling. Cells stably expressing both an optimized HAL reporter and the TCF/LEF reporter enabled bidirectional reporter activities in response to Wnt signaling. Increased HAL reporter activity and decreased TCF/LEF reporter activity were observed simultaneously in the cells when ß-catenin/TCF7L2 was inhibited. Notably, this method could decrease the number of false positives observed when screening an inhibitor library compared with the conventional TCF/LEF assay. We found that Brefeldin A, a disruptor of the Golgi apparatus, inhibited the Wnt/ß-catenin signaling pathway. The utility of our system could be expanded to examine other disease-associated pathways beyond the Wnt/ß-catenin signaling pathway.

Direct Regulation of Histidine Ammonia-Lyase 2 Gene by Thyroid Hormone in the Developing Adult Intestinal Stem Cells.

Most vertebrate organs use adult stem cells to maintain homeostasis and ensure proper repair when damaged. How such organ-specific stem cells are formed during vertebrate development is largely unexplored. We have been using the thyroid hormone (T3)-dependent amphibian metamorphosis to address this issue. Early studies in Xenopus laevis have shown that intestinal remodeling involves complete degeneration of the larval epithelium and de novo formation of adult stem cells through dedifferentiation of some larval epithelial cells. We have further discovered that the histidine ammonia-lyase (HAL; also known as histidase or histidinase)-2 gene is strongly and specifically activated by T3 in the proliferating adult stem cells of the intestine during metamorphosis, implicating a role of histidine catabolism in the development of adult intestinal stem cells. To determine the mechanism by which T3 regulates the HAL2 gene, we have carried out bioinformatics analysis and discovered a putative T3 response element (TRE) in the HAL2 gene. Importantly, we show that this TRE is bound by T3 receptor (TR) in the intestine during metamorphosis. The TRE is capable of binding to the heterodimer of TR and 9-cis retinoic acid receptor (RXR) in vitro and mediate transcriptional activation by liganded TR/RXR in frog oocytes. More importantly, the HAL2 promoter containing the TRE can drive T3-dependent reporter gene expression to mimic endogenous HAL2 expression in transgenic animals. Our results suggest that the TRE mediates the induction of HAL2 gene by T3 in the developing adult intestinal stem cells during metamorphosis.

Association of Rare Loss-Of-Function Alleles in HAL, Serum Histidine: Levels and Incident Coronary Heart Disease.

BACKGROUND: Histidine is a semiessential amino acid with antioxidant and anti-inflammatory properties. Few data are available on the associations between genetic variants, histidine levels, and incident coronary heart disease (CHD) in a population-based sample. METHODS AND RESULTS: By conducting whole exome sequencing on 1152 African Americans in the Atherosclerosis Risk in Communities (ARIC) study and focusing on loss-of-function (LoF) variants, we identified 3 novel rare LoF variants in HAL, a gene that encodes histidine ammonia-lyase in the first step of histidine catabolism. These LoF variants had large effects on blood histidine levels (ß=0.26; P=1.2×10(-13)). The positive association with histidine levels was replicated by genotyping an independent sample of 718 ARIC African Americans (minor allele frequency=1%; P=1.2×10(-4)). In addition, high blood histidine levels were associated with reduced risk of developing incident CHD with an average of 21.5 years of follow-up among African Americans (hazard ratio=0.18; P=1.9×10(-4)). This finding was validated in an independent sample of European Americans from the Framingham Heart Study (FHS) Offspring Cohort. However, LoF variants in HAL were not directly significantly associated with incident CHD after meta-analyzing results from the CHARGE Consortium. CONCLUSIONS: Three LoF mutations in HAL were associated with increased histidine levels, which in turn were shown to be inversely related to the risk of CHD among both African Americans and European Americans. Future investigations on the association between HAL gene variation and CHD are warranted.

Associations between variants of the HAL gene and milk production traits in Chinese Holstein cows.

BACKGROUND: The histidine ammonia-lyse gene (HAL) encodes the histidine ammonia-lyase, which catalyzes the first reaction of histidine catabolism. In our previous genome-wide association study in Chinese Holstein cows to identify genetic variants affecting milk production traits, a SNP (rs41647754) located 357 bp upstream of HAL, was found to be significantly associated with milk yield and milk protein yield. In addition, the HAL gene resides within the reported QTLs for milk production traits. The aims of this study were to identify genetic variants in HAL and to test the association between these variants and milk production traits. RESULTS: Fifteen SNPs were identified within the regions under study of the HAL gene, including three coding mutations, seven intronic mutations, one promoter region mutation, and four 3'UTR mutations. Nine of these identified SNPs were chosen for subsequent genotyping and association analyses. Our results showed that five SNP markers (ss974768522, ss974768525, ss974768531, ss974768533 and ss974768534) were significantly associated with one or more milk production traits. Haplotype analysis showed that two haplotype blocks were significantly associated with milk yield and milk protein yield, providing additional support for the association between HAL variants and milk production traits in dairy cows (P < 0.05). CONCLUSION: Our study shows evidence of significant associations between SNPs within the HAL gene and milk production traits in Chinese Holstein cows, indicating the potential role of HAL variants in these traits. These identified SNPs may serve as genetic markers used in genomic selection schemes to accelerate the genetic gains of milk production traits in dairy cattle.

Hepatic amino acid-degrading enzyme expression is downregulated by natural and synthetic ligands of PPARα in rats.

Body nitrogen retention is dependent on the amount of dietary protein consumed, as well as the fat and carbohydrate content in the diet, due to the modulation of amino acid oxidation. PPARα is a transcription factor involved in the upregulation of the expression of enzymes of fatty acid oxidation. However, the role of putative PPARα response elements (PPREs) in the promoter of several amino acid-degrading enzymes (AADEs) is not known. The aim of this work was to study the effect of the synthetic ligand Wy 14643 and the natural ligands palmitate, oleate, and linoleate in rats fed graded concentrations of dietary protein (6, 20, or 50 g/100 g of total diet) on the expression of the AADEs histidase, serine dehydratase, and tyrosine aminotransferase. Thus, we fed male Wistar rats diets containing 6, 20, or 50% casein for 10 d. The results showed that addition of Wy 14643 to the diet significantly reduced the expression of the AADEs. Furthermore, the incubation of hepatocytes with natural ligands of PPARα or feeding rats with diets containing soybean oil, safflower oil, lard, or coconut oil as sources of dietary fat significantly repressed the expression of the AADEs. Gene reporter assays and mobility shift assays demonstrated that the PPRE located at -482 bp of the histidase gene actively bound PPARα in rat hepatocytes. These data indicate that PPARα ligands may reduce amino acid catabolism in rats.

Neonatal blue light phototherapy and melanocytic nevi: a twin study.

BACKGROUND: Neonatal blue light phototherapy (NBLP) has been widely and successfully used for the treatment of neonatal jaundice to reduce the plasma concentration of bilirubin and, hence, to prevent kernicterus. Only a few and controversial data are available in the literature as to how NBLP influences melanocytic nevus development. OBJECTIVE: Our goal was to conduct a twin study with the aim of better understanding the role of NBLP in melanocytic nevus development. We also investigated the roles of other environmental and constitutional factors in nevus formation. METHODS: Fifty-nine monozygotic and dizygotic twins were included in this cross-sectional study. One of the twin members received NBLP, and the other did not. A whole-body skin examination was performed to determine the density of melanocytic skin lesions. The prevalence of benign pigmented uveal lesions was evaluated during a detailed ophthalmologic examination. A standardized questionnaire was used to assess data relating to constitutional, sun-exposure, and other variables. To search for possible gene-environmental interactions involved in the appearance of pigmented lesions, the melanocortin 1 receptor variants and the I439V polymorphism of histidine ammonia-lyase genes were also determined in the enrolled twins. RESULTS: NBLP was associated with a significantly higher prevalence of both cutaneous and uveal melanocytic lesions. No association was found between the examined gene polymorphisms and the number of pigmented alterations in the examined study group. CONCLUSIONS: Our data suggest that NBLP could well be a risk factor for melanocytic nevus development. Phototherapy with blue-light lamps is a standard and essential therapeutic modality in neonatal care; therefore, additional in vivo and in vitro studies are necessary to establish its potential long-term adverse effects.

Histidine protects against zinc and nickel toxicity in Caenorhabditis elegans.

Zinc is an essential trace element involved in a wide range of biological processes and human diseases. Zinc excess is deleterious, and animals require mechanisms to protect against zinc toxicity. To identify genes that modulate zinc tolerance, we performed a forward genetic screen for Caenorhabditis elegans mutants that were resistant to zinc toxicity. Here we demonstrate that mutations of the C. elegans histidine ammonia lyase (haly-1) gene promote zinc tolerance. C. elegans haly-1 encodes a protein that is homologous to vertebrate HAL, an enzyme that converts histidine to urocanic acid. haly-1 mutant animals displayed elevated levels of histidine, indicating that C. elegans HALY-1 protein is an enzyme involved in histidine catabolism. These results suggest the model that elevated histidine chelates zinc and thereby reduces zinc toxicity. Supporting this hypothesis, we demonstrated that dietary histidine promotes zinc tolerance. Nickel is another metal that binds histidine with high affinity. We demonstrated that haly-1 mutant animals are resistant to nickel toxicity and dietary histidine promotes nickel tolerance in wild-type animals. These studies identify a novel role for haly-1 and histidine in zinc metabolism and may be relevant for other animals.

Increased sensitivity of histidinemic mice to UVB radiation suggests a crucial role of endogenous urocanic acid in photoprotection.

Urocanic acid (UCA) is produced by the enzyme histidase and accumulates in the stratum corneum of the epidermis. In this study, we investigated the photoprotective role of endogenous UCA in the murine skin using histidinemic mice, in which the gene encoding histidase is mutated. Histidase was detected by immunohistochemistry in the stratum granulosum and stratum corneum of the normal murine skin but not in the histidinemic skin. The UCA content of the stratum corneum and the UVB absorption capacity of aqueous extracts from the stratum corneum were significantly reduced in histidinemic mice as compared with wild-type mice. When the shaved back skin of adult mice was irradiated with 250 mJ cm(-2) UVB, histidinemic mice accumulated significantly more DNA damage in the form of cyclobutane pyrimidine dimers than did wild-type mice. Furthermore, UVB irradiation induced significantly higher levels of markers of apoptosis in the epidermis of histidinemic mice. Topical application of UCA reversed the UVB-photosensitive phenotype of histidinemic mice and increased UVB photoprotection of wild-type mice. Taken together, these results provide strong evidence for an important contribution of endogenous UCA to the protection of the epidermis against the damaging effects of UVB radiation.

A role for ultraviolet radiation immunosuppression in non-melanoma skin cancer as evidenced by gene-environment interactions.

The genotoxic effects of ultraviolet (UV) radiation are well-known causes of skin cancers; however, UV radiation also suppresses the immune system, decreasing the body's surveillance for tumor cells. In experimental systems, UV radiation immunosuppression is at least partially mediated through urocanic acid (UCA), an UV radiation-absorbing molecule in the stratum corneum. We tested the hypothesis that genetic variation in the histidase gene (HAL), which catalyzes the formation of UCA in the skin, modifies risk of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) in a population-based study (914 BCC, 702 SCC and 848 controls). We observed no evidence of a main gene effect for the HAL I439V polymorphism (rs7297245) and BCC or SCC. However, we found a HAL genotype-sunburn interaction in association with BCC (P for interaction = 0.040) and SCC (P for interaction = 0.018). A HAL genotype-SCC association was observed primarily among women (odds ratio = 1.5, 95% confidence interval 1.1-2.2), and among women, we found an interaction between HAL genotype and oral contraceptive use on SCC risk (P = 0.040). The variant HAL allele likewise appeared to modify the SCC risk associated with glucocorticoid steroid usage (P for interaction = 0.0004). In conclusion, our findings are a first step in determining the genetic underpinnings of UV immune suppression and have identified important new genetic interactions contributing to the etiology of skin cancer.

Histidase expression in human epidermal keratinocytes: regulation by differentiation status and all-trans retinoic acid.

BACKGROUND: Histidase (histidine ammonia lyase) converts histidine into urocanic acid, the main ultraviolet (UV) light absorption factor of the stratum corneum. It is unknown if and how histidase is regulated in the epidermis. OBJECTIVE: We have investigated the transcriptional regulation of histidase expression in epidermal keratinocytes. METHODS: Human epidermal keratinocytes were cultured in vitro and exposed to UV irradiation, a number of cytokines and all-trans retinoic acid (ATRA) (1 microM). Keratinocyte differentiation was triggered by maintaining confluent cells in monolayer culture and by establishing three-dimensional skin equivalents. The mRNA expression level of histidase in keratinoytes as well as in the epidermis and other tissues was determined by quantitative real-time PCR. Protein expression was determined by Western blot analysis. RESULTS: Human epidermis contained higher levels of histidase transcripts than all other tissues investigated. Expression of histidase strongly increased at the mRNA and protein levels during differentiation of primary keratinocytes in vitro. Treatment of keratinocytes with UVA and UVB did not significantly change the expression level of histidase. By contrast, ATRA suppressed histidase expression almost completely. CONCLUSIONS: Our results show that histidase is upregulated during keratinocyte differentiation and that ATRA but not UV irradiation modulates the expression level of histidase. Suppression of histidase-mediated production of urocanic acid may contribute to the increase in UV sensitivity that is caused by treatment with retinoids.

Dietary nitrogen intake regulates hepatic malic enzyme messenger ribonucleic acid expression.

Increased dietary protein intake rapidly (3 h) decreases malic enzyme and increases hepatic histidase mRNA expression. Experiments were conducted to determine the role that individual dispensable amino acids and nonprotein N sources might have in regulating the activity of these enzymes and to determine if the addition of a N supplement to a practical broiler diet during the entire rearing period would reduce abdominal fat accumulation in broilers. Broiler chicks were fed a basal diet containing 22% protein or this diet supplemented with 9.5% l-Glu, 5% Gly, 6% l-Ala, 5.08% ammonium bicarbonate, or 4.25% dibasic ammonium phosphate for 24 h. Each of the dietary supplements added 0.90% total N to the diet. Hepatic malic enzyme mRNA expression was significantly (P < 0.05) depressed in chicks fed any of the supplemented diets compared with chicks fed the basal diet. Histidase mRNA expression, however, was only significantly increased in the chicks fed the basal diet supplemented with Gly. Broilers fed practical corn-soybean meal starter and developer diets supplemented with 2.3, 4.7, or 9.5% Glu from 0 to 40 d of age had significantly smaller abdominal fat pads relative to BW than broilers fed the unsupplemented corn-soybean meal diets. Feeding the Glu supplements, however, reduced the overall BW gain of broilers by 100 to 150 g compared with broilers fed the unsupplemented diets. The results suggest that hepatic mRNA expression of malic enzyme may be regulated by total dietary N intake, whereas hepatic mRNA expression of histidase may be regulated by specific amino acids.

Fasting-induced changes in ECL cell gene expression.

Gastric enterochromaffin-like (ECL) cells release histamine in response to food because of elevation of gastrin and neural release of pituitary adenylate cyclase-activating peptide (PACAP). Acid secretion is at a basal level in the absence of food but is rapidly stimulated with feeding. Rats fasted for 24 h showed a significant decrease of mucosal histamine despite steady-state expression of the histamine-synthesizing enzyme histidine decarboxylase (HDC). Comparative transcriptomal analysis using gene expression oligonucleotide microarrays of 95% pure ECL cells from fed and 24-h fasted rats, thereby eliminating mRNA contamination from other gastric mucosal cell types, identified significantly increased gene expression of the enzymes histidase and urocanase catabolizing the HDC substrate L-histidine but significantly decreased expression of the cellular L-histidine uptake transporter SN2 and of the vesicular monoamine transporter 2 (VMAT-2) responsible for histamine uptake into secretory vesicles. This was confirmed by reverse transcriptase-quantitative polymerase chain reaction of gastric fundic mucosal samples from fed and 24-h fasted rats. The decrease of VMAT-2 gene expression was also shown by a decrease in VMAT-2 protein content in protein extracts from fed and 24-h fasted rats compared with equal amounts of HDC protein and Na-K-ATPase alpha(1)-subunit protein content. These results indicate that rat gastric ECL cells regulate their histamine content during 24-h fasting not by a change in HDC gene or protein expression but by regulation of substrate concentration for HDC and a decreased histamine secretory pool.

The role of glucagon in regulating chicken hepatic malic enzyme and histidase messenger ribonucleic acid expression in response to an increase in dietary protein intake.

Increased dietary protein intake rapidly (3 h) decreases hepatic malic enzyme and increases hepatic histidase mRNA expression in broiler chicks. A series of experiments was conducted to determine the role that glucagon or a specific mixture of dietary amino acids might have in regulating the rapid changes in mRNA expression of these enzymes, when dietary protein intake is increased. Three hours after the injection of glucagon (240 microg/kg of BW) into the brachial vein of broiler chicks, hepatic malic enzyme mRNA expression was significantly lower and hepatic histidase mRNA expression was significantly greater than the level detected in saline-injected chicks. In addition, broiler chicks fed a high (40 g/ 100 g of diet) protein diet had significantly higher plasma glucagon levels at 1 and 3 h after initial access to this diet than broiler chicks fed a basal (22 g/100 g of diet) protein diet. The plasma glucagon concentration, however, was not different between the chicks fed the 2 dietary protein levels at 2 h after the initial access to the 2 diets. When a mixture of indispensable or dispensable amino acids was added to the basal diet to equal the concentrations of the individual indispensable or dispensable amino acids in the high protein diet, hepatic mRNA expression of malic enzyme and histidase were intermediate to the expression found in chicks fed the basal and high protein diet. The results indicate that glucagon may mediate the changes in the mRNA expression of malic enzyme and histidase in response to dietary protein intake and that total amino acid intake rather than the ingestion of specific amino acids regulates the mRNA expression of malic enzyme and histidase in chicks.

The essential tyrosine-containing loop conformation and the role of the C-terminal multi-helix region in eukaryotic phenylalanine ammonia-lyases.

Besides the post-translationally cyclizing catalytic Ala-Ser-Gly triad, Tyr110 and its equivalents are of the most conserved residues in the active site of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), histidine ammonia-lyase (HAL, EC 4.3.1.3) and other related enzymes. The Tyr110Phe mutation results in the most pronounced inactivation of PAL indicating the importance of this residue. The recently published X-ray structures of PAL revealed that the Tyr110-loop was either missing (for Rhodospridium toruloides) or far from the active site (for Petroselinum crispum). In bacterial HAL ( approximately 500 amino acids) and plant and fungal PALs ( approximately 710 amino acids), a core PAL/HAL domain ( approximately 480 amino acids) with >or= 30% sequence identity along the different species is common. In plant and fungal PAL a approximately 100-residue long C-terminal multi-helix domain is present. The ancestor bacterial HAL is thermostable and, in all of its known X-ray structures, a Tyr83-loop-in arrangement has been found. Based on the HAL structures, a Tyr110-loop-in conformation of the P. crispum PAL structure was constructed by partial homology modeling, and the static and dynamic behavior of the loop-in/loop-out structures were compared. To study the role of the C-terminal multi-helix domain, Tyr-loop-in/loop-out model structures of two bacterial PALs (Streptomyces maritimus, 523 amino acids and Photorhabdus luminescens, 532 amino acids) lacking this C-terminal domain were also built. Molecular dynamics studies indicated that the Tyr-loop-in conformation was more rigid without the C-terminal multi-helix domain. On this basis it is hypothesized that a role of this C-terminal extension is to decrease the lifetime of eukaryotic PAL by destabilization, which might be important for the rapid responses in the regulation of phenylpropanoid biosynthesis.