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PubMed1999

Use of molecular and isotopic techniques to monitor the response of autotrophic ammonia-oxidizing populations of the beta subdivision of the class proteobacteria in arable soils to nitrogen fertilizer.

This study examined the effects of NH(4)NO(3) fertilizer on the size and activity of nitrifying, autotrophic, ammonia-oxidizing populations of the beta subdivision of the class Proteobacteria in arable soils. Plots under different long-term fertilizer regimes were sampled before and after NH(4)NO(3) additions, and the rates of nitrification were determined by (15)N isotopic pool dilution assays. Ammonia-oxidizing populations in the plots were quantified by competitive PCR assays based on the amoA and ribosomal 16S genes. Prior to fertilizer addition, ammonium concentrations and nitrification rates in the plots were comparatively low; ammonia-oxidizing populations were present at 10(4) to 10(5) gene copies g of soil(-1). Three days after the application of fertilizer, nitrification rates had risen considerably but the size of the ammonia-oxidizing population was unchanged. Six weeks after fertilizer treatment, ammonium concentrations and nitrification rates had fallen while the ammonia-oxidizing populations in plots receiving fertilizer had increased. The rapidity of the rise in nitrification rates observed after 3 days suggests that it results from phenotypic changes in the ammonia-oxidizing bacterial population. Associated increases in population sizes were only observed after 6 weeks and did not correlate directly with nitrifying activity. Phylogenetic analyses of PCR products from one of the plots revealed a population dominated by Nitrosospira-type organisms, similar to those prevalent in other soils.

Betaproteobacteria↗
PubMed1993

Sequence of the gene coding for ammonia monooxygenase in Nitrosomonas europaea.

Nitrosomonas europaea, a chemolithotrophic bacterium, was found to contain two copies of the gene coding for the presumed active site polypeptide of ammonia monooxygenase, the 32-kDa acetylene-binding polypeptide. One copy of this gene was cloned, and its complete nucleotide sequence is presented. Immediately downstream of this gene, in the same operon, is the gene for a 40-kDa polypeptide that copurifies with the ammonia monooxygenase acetylene-binding polypeptide. The sequence of the first 692 nucleotides of this structural gene, coding for about two-thirds of the protein, is presented. These sequences are the first sequences of protein-encoding genes from an ammonia-oxidizing autotrophic nitrifying bacterium. The two protein sequences are not homologous with the sequences of any other monooxygenase. From radioactive labelling of ammonia monooxygenase with [14C]acetylene it was determined that there are 23 nmol of ammonia monooxygenase per g of cells. The kcat of ammonia monooxygenase for NH3 in vivo was calculated to be 20 s-1.

Acetylene↗
PubMed2000

Modification of the epidermal growth factor response by ammonia in renal cell hypertrophy.

Epidermal growth factor (EGF) causes proliferation in renal tubular cells but, when it is combined with transforming growth factor-beta1, it causes hypertrophy by a mechanism that requires the activity of the retinoblastoma family of proteins. In contrast, ammonia causes hypertrophy by decreasing lysosomal proteolysis; in some cell types, it also decreases cellular proliferation. These studies were designed to determine whether ammonia, like transforming growth factor-beta1, could convert EGF-induced hyperplasia to hypertrophy. Cultured NRK-52E cells were incubated with EGF and/or ammonia and the protein/DNA ratio was measured, as a marker of hypertrophy. Addition of ammonia to EGF-treated NRK-52E cells converted EGF-induced hyperplasia to hypertrophy, because of a decrease in DNA synthesis. The mechanism involved no change in EGF-induced protein synthesis. Inhibition of lysosomal function with a proton pump inhibitor or lysosomal protease inhibitors also converted the response of EGF-treated cells to hypertrophy. Expression of the human papilloma virus 16 E7 protein (which inactivates all members of the retinoblastoma family) prevented ammonia from converting EGF-induced hyperplasia to hypertrophy. It is concluded that ammonia converts EGF-induced hyperplasia to hypertrophy by a mechanism that involves suppression of lysosomal function and this response can be blocked by inhibiting the activity of the retinoblastoma family of proteins.

Alkalies↗
PubMed1979

Gene expression in Dictyostelium discoidium: mutually antagonistic roles of cyclic-AMP and ammonia.

Cyclic-AMP and ammonia have been previously identified as extracellular signals during Dictyostelium development. Both are important in controlling morphological movements and cyclic-AMP also in inducing gene expression. The work in tis paper is concerned with their effects on developmental gene expression. Cyclic-AMP was found to act as an inducer during the aggregative (as exemplified by phosphodiesterase) and the post-aggregative (glycogen phosphorylase, UDP-galactose polysaccharide transferase, prespore vacuoles and stalk cells) phases of gene expression. Ammonia inhibited the appearance of each of the above markers and antagonized the inductive effects of cyclic-AMP on them. This inhibition by ammonia of cyclic-AMP inducible gene expression may involve a step linking elevated intracellular cyclic-AMP levels to gene activation. It has been suggested that the specification of cells within the aggregate into the stalk and spore pathways of differentiation might be controlled by cyclic-AMP and ammonia. In this model for pattern formation cyclic-AMP would induce stalk cell differentiation and ammonia spore formation. The present results argue against this idea since cyclic-AMP induces and ammonia inhibits differentiation along both pathways. The function of these agents may rather be to coordinate the rates of biochemical differentiation of individual cells and link them to the overall morphological changes occurring during development.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1988

Stimulation of alanine metabolism in rat liver by ammonia.

The effect of ammonia on the alanine metabolism was investigated in perfused rat liver. Gluconeogenesis was found to be stimulated by physiological concentrations of ammonia, while being inhibited at higher concentrations (5-10 mM). The stimulating effect of 0.5 mM ammonia was studied in greater detail. In addition to glucose formation seen enhanced five times, increased rates were observed for ureogenesis as well as the formation of lactate and pyruvate, demonstrating also activation of the total alanine turnover. Furthermore, the mitochondrial and cytosolic NAD systems were increasingly oxidized as reflected by the beta-hydroxybutyrate/acetoacetate and lactate/pyruvate ratios. The shift of the beta-hydroxybutyrate/acetoacetate ratio was correlated to the ATP demand by gluconeogenesis and ureogenesis. The elevated concentration of pyruvate was found to have caused stimulation of gluconeogenesis since there existed a Michaelis-Menten type relation between pyruvate concentration and glucose formation irrespective of the presence or absence of ammonia. The flux through glutamate dehydrogenase was calculated from the total alanine turnover and urea formation, and noted to be diminished in the presence of ammonia despite the increased alanine turnover. It is concluded that glutamate dehydrogenase, at least in part, controls the total alanine turnover in the absence of ammonia.

Alanine↗
PubMed2005

Limited capacity for ammonia removal by brain in chronic liver failure: potential role of nitric oxide.

Chronic liver failure leads to hyperammonemia and consequently increased brain ammonia concentrations, resulting in hepatic encephalopathy. When the liver fails to regulate ammonia concentrations, the brain, devoid of a urea cycle, relies solely on the amidation of glutamate to glutamine through glutamine synthetase, to efficiently clear ammonia. Surprisingly, under hyperammonemic conditions, the brain is not capable of increasing its capacity to remove ammonia, which even decreases in some regions of the brain. This non-induction of glutamine synthetase in astrocytes could result from possible limiting substrates or cofactors for the enzyme, or an indirect effect of ammonia on glutamine synthetase expression. In addition, there is evidence that nitration of the enzyme resulting from exposure to nitric oxide could also be implicated. The present review summarizes these possible factors involved in limiting the increase in capacity of glutamine synthetase in brain, in chronic liver failure.

Ammonia↗
PubMed1987

A rapid assay method for ammonia using glutamine synthetase from glutamate-producing bacteria.

A rapid enzymatic assay method for ammonia was developed by using glutamine synthetase from glutamate-producing bacteria together with pyruvate kinase, lactate dehydrogenase, and NADH. The time required for determination of 25 nmol of ammonia was 5 min with 1 unit of glutamine synthetase, as opposed to 14-30 min with 1 unit of glutamate dehydrogenases from various sources. The present method was used to determine ammonia in serum, microbiol-culture broth, and waste water. The method can be modified for spectrophotometry in the visible region by substituting pyruvate oxidase, peroxidase, and appropriate chromogens for lactate dehydrogenase and NADH. With 4-aminoantipyrine (4AA) and phenol, and with 4AA and N-ethyl-N-2-hydroxyethyl-m-toluidine as chromogens, the sensitivity of ammonia determination was 0.65 and 1.7 times that with glutamate dehydrogenase, respectively. The present method was also applicable to the continuous detection of the activity of some ammonia-forming enzymes such as guanase, adenosine deaminase, and urease and to the determination of 0.5-30 microM ATP-ADP after some modification of the mixture.

Adenosine Diphosphate↗
PubMed2001

Mixture of N-carbamoyl-L-glutamate plus L-arginine can protect rats with liver cirrhosis from acute ammonia intoxication.

BACKGROUND/AIMS: We earlier reported that N-carbamoyl-L-glutamate (CG) plus L-arginine (Arg) protected normal and 70% hepatectomized rats from intoxication by a lethal or sub-lethal dose of ammonium acetate, respectively. In the present study, the protective effect of these compounds on cirrhotic rats was assessed. METHODS: CG plus Arg were administered prior to the injection of a sub-lethal dose of ammonium acetate into dimethylnitrosamine-induced cirrhotic rats. Control rats were given phosphate-buffered saline (PBS) instead of the mixture. The behavior of the rats was monitored until the time of sacrifice. Blood ammonia level, blood urea nitrogen (BUN) and liver carbamoylphosphate synthetase I (CPS I) activity were determined. RESULTS: Pretreatment of rats with the mixture of CG plus Arg could significantly lower the blood ammonia level (P<0.05), increase the activity of CPS I (P<0.05), improve abnormal behavior associated with ammonia intoxication (P<0.05), and increase BUN (P<0.05), as compared with the PBS-injected control group. There were significantly close correlations between (1) the increase of CPS I activity; (2) the improvement of abnormal behavior; (3) the increase of BUN; and (4) the decrease of the blood ammonia level. CONCLUSIONS: A mixture of CG plus Arg could protect rats with liver cirrhosis from acute ammonia intoxication.

Acetates↗
PubMedDate not supplied

Glutamine synthetase in brain: effect of ammonia.

Glutamine synthetase (GS) in brain is located mainly in astrocytes. One of the primary roles of astrocytes is to protect neurons against excitotoxicity by taking up excess ammonia and glutamate and converting it into glutamine via the enzyme GS. Changes in GS expression may reflect changes in astroglial function, which can affect neuronal functions. Hyperammonemia is an important factor responsible of hepatic encephalopathy (HE) and causes astroglial swelling. Hyperammonemia can be experimentally induced and an adaptive astroglial response to high levels of ammonia and glutamate seems to occur in long-term studies. In hyperammonemic states, astroglial cells can experience morphological changes that may alter different astrocyte functions, such as protein synthesis or neurotransmitters uptake. One of the observed changes is the increase in the GS expression in astrocytes located in glutamatergic areas. The induction of GS expression in these specific areas would balance the increased ammonia and glutamate uptake and protect against neuronal degeneration, whereas, decrease of GS expression in non-glutamatergic areas could disrupt the neuron-glial metabolic interactions as a consequence of hyperammonemia. Induction of GS has been described in astrocytes in response to the action of glutamate on active glutamate receptors. The over-stimulation of glutamate receptors may also favour nitric oxide (NO) formation by activation of NO synthase (NOS), and NO has been implicated in the pathogenesis of several CNS diseases. Hyperammonemia could induce the formation of inducible NOS in astroglial cells, with the consequent NO formation, deactivation of GS and dawn-regulation of glutamate uptake. However, in glutamatergic areas, the distribution of both glial glutamate receptors and glial glutamate transporters parallels the GS location, suggesting a functional coupling between glutamate uptake and degradation by glutamate transporters and GS to attenuate brain injury in these areas. In hyperammonemia, the astroglial cells located in proximity to blood-vessels in glutamatergic areas show increased GS protein content in their perivascular processes. Since ammonia freely crosses the blood-brain barrier (BBB) and astrocytes are responsible for maintaining the BBB, the presence of GS in the perivascular processes could produce a rapid glutamine synthesis to be released into blood. It could, therefore, prevent the entry of high amounts of ammonia from circulation to attenuate neurotoxicity. The changes in the distribution of this critical enzyme suggests that the glutamate-glutamine cycle may be differentially impaired in hyperammonemic states.

Amino Acid Transport System X-AG↗
PubMed2026

Adaptive Evolution Reveals Metabolic Plasticity and Functional Redundancy in an Anaerobic Microbiome under Extreme Ammonia Stress.

Ammonia toxicity represents a primary biochemical bottleneck governing microbial community structure and performance during the anaerobic digestion of the organic fraction of municipal solid waste. However, the mechanistic basis of microbial adaptation to chronic ammonia levels remains poorly characterized. In this study, a long-term sequential enrichment strategy under progressively increasing ammonia concentrations (350-1500 mgN L-1), integrated with genome-centric metagenomics and metatranscriptomics, was employed to resolve the response of an organic waste-degrading microbiome over a 240 day period. Increasing ammonia pressure induced a progressive decline in methanogenesis and accumulation of volatile fatty acids, particularly acetate. Despite these inhibitory pressures, methane production was only halved relative to the initial baseline reflecting a resilient methanogenic community. This stability was driven by a restructuring of the microbiome, where functional redundancy across divergent taxa preserved core metabolic functions. Key adaptive responses included the reconfiguration of carbon fixation pathways, specifically via a variant of the Wood-Ljungdahl pathway coupled with the glycine cleavage system acting as an alternative acetate oxidation route, as well as sustained osmoprotectant biosynthesis. Cellular homeostasis was preserved through H+ replenishment via multiple energy-converting complexes and K+ influx to maintain cation-proton balance. Collectively, these findings demonstrate that metabolic plasticity and the preservation of core metabolic functions are the primary determinants of ammonia resilience, sustaining methane production under inhibitory conditions.

Ammonia↗
PubMed1990

Wound-induced phenylalanine ammonia-lyase in potato (Solanum tuberosum) tuber discs. Significance of glycosylation and immunolocalization of enzyme subunits.

1. Excised discs of potato (Solanum tuberosum) tuber were incubated with [3H]fucose and extracts were prepared and incubated with an antibody to phenylalanine ammonia-lyase. Analysis of the resulting immunoprecipitated proteins by SDS/PAGE showed [3H]mannose- and [3H]fucose-labelled bands with Mr values corresponding to those of phenylalanine ammonia-lyase subunits. 2. When potato discs were incubated with [3H]sugars in the presence of tunicamycin, an inhibitor of N-linked protein glycosylation, incorporation of radioactivity from [3H]mannose into the immunoprecipitated enzyme subunits was virtually eliminated, whereas that from [3H]fucose was only marginally inhibited. 3. Tunicamycin reduced the level of extractable phenylalanine ammonia-lyase activity induced in excised potato tuber discs. Kinetic analysis revealed that the Vmax value of the enzyme in crude extracts from tunicamycin-treated tissue was reduced, whereas the apparent Km values were unaffected. 4. Immunoprecipitation of the enzyme labelled in vivo with [35S]methionine showed that tunicamycin did not inhibit the synthesis of the enzyme protein per se, nor did it increase the degradation of the enzyme protein. 5. Immunoprecipitation of the enzyme labelled in vitro with [14C]nitromethane showed that tunicamycin did not affect the introduction of the dehydroalanine residue into the active site. 6. These results are consistent with the following hypothesis: tunicamycin inhibits the N-linked glycosylation of phenylalanine ammonia-lyase which, in turn, results in imperfect folding of the enzyme protein. The orientation of the active site is changed in such a way that the affinity of the enzyme for its substrate is unaffected, whereas the catalytic activity of the enzyme is reduced. 7. Both optical- and electron-microscopic immunolocalization studies with antibody to phenylalanine ammonia-lyase showed increased deposition of silver granules in cells in sections of potato discs in which induction of the enzyme was allowed to occur compared with cells from newly wounded tissue. The enzyme was located in the cytoplasm, and was possibly membrane-associated.

Ammonia-Lyases↗
PubMed1983

Rapid induction of the synthesis of phenylalanine ammonia-lyase and of chalcone synthase in elicitor-treated plant cells.

Changes in the rate of synthesis of phenylalanine ammonia-lyase and chalcone synthase, two characteristic enzymes of phenylpropanoid biosynthesis, have been investigated by direct immunoprecipitation of in vivo [35S]methionine-labelled enzyme subunits in elicitor-treated cells of dwarf French bean (Phaseolus vulgaris). Elicitor, heat-released from cell walls of Colletotrichum lindemuthianum, the causal agent of anthracnose disease of bean, causes marked but transient increases in the rates of synthesis of both enzymes concomitant with the phase of rapid increase in enzyme activity at the onset of phaseollin accumulation during the phytoalexin defence response. Increased rates of synthesis of both enzymes can be observed 20 min after elicitor treatment and the pattern of induction of synthesis of phenylalanine ammonia-lyase and chalcone synthase are broadly similar with respect to elicitor concentration and time, maximum rates of synthesis being attained between 2.5 h and 3.0 h after elicitor treatment. Within this overall co-ordination small but distinct differences between the enzymes were observed in: (a) the elicitor concentrations giving maximum enzyme synthesis, and (b) the precise timing of maximum enzyme synthesis, with that for chalcone synthase occurring 20-30 min earlier than that for phenylalanine ammonia-lyase. However, for a given rate of enzyme synthesis, induction of the activities of phenylalanine ammonia-lyase and chalcone synthase is more efficient at high elicitor concentrations. This may reflect the operation under certain circumstances of post-translational control of the activity levels of these enzymes as implicated for phenylalanine ammonia-lyase by previous density-labelling experiments [Lawton et al. (1980) Biochim. Biophys. Acta, 633, 162-175]. The same pattern of induction of enzyme synthesis is observed with elicitor preparations from a variety of sources.

Acyltransferases↗
PubMed1988

Purification, characterization and induction of L-phenylalanine ammonia-lyase in Phaseolus vulgaris.

The enzyme L-phenylalanine ammonia-lyase was purified from leaves of Phaseolus vulgaris by Sephacryl S-200 gel filtration and Sepharose-4-B--succinyl-aminoethyl-L-phenylalanine affinity chromatography. L-Phenylalanine ammonia-lyase was specifically eluted from the affinity matrix with its substrate L-phenylalanine at 20-25 degrees C. The purified enzyme was shown to be homogeneous by gel electrophoresis both in presence and absence of SDS. Its Mr, determined by gel filtration and non-denaturing gel electrophoresis, was 320,000 +/- 9000 and 330,000 +/- 4000 respectively. After SDS electrophoresis only one band of Mr 83,000 +/- 4000 was detected, indicating that the enzyme is an oligomer containing four subunits. The pH optimum of enzyme activity was 8.8-9.2. Ampholyte isoelectrofocusing in polyacrylamide demonstrated the presence of a single charged species at pH 4.2. The homogeneous enzyme catalyzed the deamination of L-phenylalanine to trans-cinnamate but did not catalyze the transamination of L-phenylalanine to L-phenylpyruvate. The enzyme showed Km 1.25 mM for L-phenylalanine. Antibodies to homogeneous L-phenylalanine ammonia-lyase recognised specific epitopes on L-phenylalanine aminotransferase as demonstrated by immunoaffinity purification and immunoblotting. The induction of L-phenylalanine ammonia-lyase activity during phaseollin biosynthesis in the Phaseolus vulgaris--Colletotrichum lindemuthianum interaction was regulated by an increase in enzyme concentration resulting from an increase in de novo synthesis of L-phenylalanine ammonia-lyase protein.

Ammonia-Lyases↗
PubMed1985

Cerebral ammonia metabolism in hyperammonemic rats.

The short-term metabolic fate of blood-borne [13N]ammonia was determined in the brains of chronically (8- or 14-week portacaval-shunted rats) or acutely (urease-treated) hyperammonemic rats. Using a "freeze-blowing" technique it was shown that the overwhelming route for metabolism of blood-borne [13N]ammonia in normal, chronically hyperammonemic and acutely hyperammonemic rat brain was incorporation into glutamine (amide). However, the rate of turnover of [13N]ammonia to L-[amide-13N]glutamine was slower in the hyperammonemic rat brain than in the normal rat brain. The activities of several enzymes involved in cerebral ammonia and glutamate metabolism were also measured in the brains of 14-week portacaval-shunted rats. The rat brain appears to have little capacity to adapt to chronic hyperammonemia because there were no differences in activity compared with those of weight-matched controls for the following brain enzymes involved in glutamate/ammonia metabolism: glutamine synthetase, glutamate dehydrogenase, aspartate aminotransferase, glutamine transaminase, glutaminase, and glutamate decarboxylase. The present findings are discussed in the context of the known deleterious effects on the CNS of high ammonia levels in a variety of diseases.

Adenosine Triphosphate↗
PubMed2026

Potential survival strategies of novel comammox and nitrite-oxidizing Nitrospira synthesizing osmoprotectants in a wastewater microbiome treating high-ammonia brackish landfill leachate.

BACKGROUND: In the late stages of landfill operation, leachate becomes brackish and contains high concentrations of ammonia with limited organic carbon. At leachate treatment facilities, it is typically subjected to nitrification followed by denitrification, with methanol supplied as an external electron donor. This unique environment may harbor novel microorganisms, including nitrifiers. Although a variety of microorganisms are involved in nitrification, their substrate specificity and salinity tolerance remain insufficiently understood. In this study, a genome-centric metagenome analysis was conducted on the microbiome from a leachate treatment facility at a closed landfill. RESULTS: A total of 68 metagenome-assembled genomes (MAGs) were reconstructed, including 64 putative novel species. Among these, two Nitrospira MAGs were recovered: a novel complete ammonia-oxidizing bacterium (comammox), Nitrospira LAS72 (88.72% completeness, 2.10% contamination), and canonical nitrite-oxidizing Nitrospira LAS18 (99.98% completeness, 2.29% contamination). Comparative genomic analysis with 260 publicly available Nitrospira genomes revealed that LAS18 represents a new sub-lineage within lineage VII of the Nitrospira genus. Two ammonia-oxidizing archaea (AOA), Candidatus Nitrosocosmicus LAS21 and Nitrosarchaeum LAS73, were also identified, while canonical ammonia-oxidizing bacteria were not detected. Given the brackish conditions (1.23% salinity) and the methanol-fed operation of the treatment facility, the genomic potential for osmotic stress adaptation and methanol metabolism was investigated. Comammox Nitrospira LAS72 harbors biosynthetic pathways for several compatible solutes (osmoprotectants), including glycine betaine, proline, trehalose, and L-glutamate. Moreover, comammox Nitrospira LAS72 possesses genetic potential for oxidizing formaldehyde, suggesting that it may exploit these methanol-derived intermediates as energy sources. These features indicate that LAS72 may withstand osmotic fluctuations through the production of various osmoprotectants and thrive under the unique conditions of a methanol-fed environment. CONCLUSIONS: The discovery of novel comammox Nitrospira and canonical Nitrospira forming a new sub-lineage within lineage VII of the Nitrospira genus in an ammonia-rich brackish environment provides the first genomic evidence for evolutionary adaptation among nitrifiers to saline, methanol-fed environments. These findings enhance our understanding of the ecological and evolutionary dynamics shaping nitrifier communities in complex treatment ecosystems. Video Abstract.

Ammonia↗
PubMed2004

Biochemical responses of two erythrinidae fish to environmental ammonia.

The non-ionized form of ammonia is very toxic to many aquatic species. It is especially important in several aspects of fish biology. A large range of organismal strategies for coping with environmental stressors is usually observed in living organisms. Among those, the responses for managing chemical stressors are well studied. The present work compares biochemical responses of two evolutionarily close species, Hoplias malabaricus and Hoplerythrinus unitaeniatus, exposed to environmental ammonia. Adult fish were submitted to 1.0 mg/L of ammonium chloride for 24 hours, and plasma ammonia and urea levels were determined. The activities of OUC enzymes OCT and ARG, and the accessory enzyme GS, were quantified in liver extract and are expressed below in mumol/min/mg of wet tissue. Increases in OUC enzymes (GS from 1.14 to 2.43, OCT from 0.81 to 1.72, and ARG from 3.15 to 4.23), plasma ammonia (from 0.95 to 1.42 mmol/L), and plasma urea (from 0.82 to 1.53 mmol/L) were observed (p < 0.05) in H. malabaricus exposed to 1 mg/L of ammonia chloride. The GS in H. unitaeniatus increased from 1.43 to 1.84, however the OCT, ARG, and plasma urea from H. unitaeniatus did not change. These data indicate that each species responds differently to the same environmental stressor.

Adaptation, Physiological↗
PubMed1990

Cloning and expression of rat histidase. Homology to two bacterial histidases and four phenylalanine ammonia-lyases.

Histidase (histidine ammonia-lyase, EC 4.3.1.3) catalyzes the deamination of histidine to urocanic acid. Apart from phenylalanine ammonia-lyase, which is not expressed in animals, histidase is the only enzyme known to have a dehydroalanine residue in its active site. The amino site precursor and the mechanism of formation of dehydroalanine are not known. As an initial step to determining the precursor of dehydroalanine in histidase, we have isolated a functional cDNA clone for histidase from a rat liver cDNA library using an affinity-purified antiserum. The 2.2-kilobase cDNA has a 1,971-base pair open reading frame coding for a 657-amino acid polypeptide with a predicted molecular mass of 72,165 Da. The cDNA has a rare polyadenylation signal (AAUACA) that appears to inefficiently direct polyadenylation in transfected COS monkey kidney cells. Conversion of this sequence to the consensus polyadenylation signal (AAUAAA) resulted in increased levels of stable mRNA. COS cells transfected with a histidase expression vector produce active histidase. The formation of active histidase in cells that have no endogenous histidase activity suggests either that the requisite modifying enzyme is present in these cells or that the dehydroalanine residue forms by an autocatalytic mechanism. Rat histidase was found to have 41 and 43% amino acid identity to Pseudomonas putida and Bacillus subtilis histidases, respectively. Phenylalanine ammonia-lyases from parsley, kidney bean, and two yeast strains were also found to have approximately 20% amino acid identity to rat histidase. On the basis of the similarity of function of histidase and phenylalanine ammonia-lyase, dehydroalanine at the active sites, and the sequence conservation over a large evolutionary distance (mammals, bacteria, yeast, and plants), we propose that the genes for histidase and phenylalanine ammonia-lyase have diverged from a common ancestral gene, of which the most conserved regions are likely to be involved in catalysis or dehydroalanine formation.

Amino Acid Sequence↗
PubMed1990

Cloning, sequencing, and expression of the genes encoding the adenosylcobalamin-dependent ethanolamine ammonia-lyase of Salmonella typhimurium.

Ethanolamine ammonia-lyase is a bacterial enzyme that catalyzes the adenosylcobalamin-dependent conversion of certain vicinal amino alcohols to oxo compounds and ammonia. Studies of ethanolamine ammonia-lyase from Clostridium sp. and Escherichia coli have suggested that the enzyme is a heterodimer composed of subunits of Mr approximately 55,000 and 35,000. Using a partial Sau3A Salmonella typhimurium library ligated into pBR328 and selecting by complementation of a mutant lacking ethanolamine ammonia-lyase activity, we have cloned the genes for the 2 subunits of the S. typhimurium enzyme. The genes were localized to a 6.5-kilobase fragment of S. typhimurium DNA, from which they could be expressed in E. coli under noninducing conditions. Sequencing of a 2526-base pair portion of this 6.5-kilobase DNA fragment revealed two open reading frames separated by 21 base pairs. The open reading frames encoded proteins of 452 and 286 residues whose derived N-terminal sequences were identical to the N-terminal sequences of the 2 subunits of the E. coli ethanolamine ammonia-lyase, except that residue 16 of the large subunit was asparagine in the E. coli sequence and aspartic acid in the S. typhimurium sequence.

Amino Acid Sequence↗