Comparative aspects of adenylic acid deaminase and aspartate-2-oxoglutarate aminotransferase.

1. The content of adenylic acid deaminase and of aspartate-2-oxoglutarate aminotransferase of skeletal muscle tissue from a variety of animals has been determined. 2. White (fast) muscle contained large amounts of adenylic acid deaminase and red (slow) muscle contained large amounts of aspartate aminotransferase. There was a general inverse relationship between the adenylic acid deaminase and the aspartate aminotransferase content of muscles from various vertebrates. Thus, there is no simple correlation between the capacity to produce inosinic acid and ammonia from adenylic acid and the capacity to catalyse the formation of aspartate for conversion of inosinic acid back to adenylic acid. 3. The absence of adenylic acid deaminase from the tail muscles of the yabbie and other invertebrates indicates a marked difference in the Animal Kingdom.     

Proteus mirabilis amino acid deaminase: cloning, nucleotide sequence, and characterization of aad.

Proteus, Providencia, and Morganella species produce deaminases that generate alpha-keto acids from amino acids. The alpha-keto acid products are detected by the formation of colored iron complexes, raising the possibility that the enzyme functions to secure iron for these species, which do not produce traditional siderophores. A gene encoding an amino acid deaminase of uropathogenic Proteus mirabilis was identified by screening a genomic library hosted in Escherichia coli DH5 alpha for amino acid deaminase activity. The deaminase gene, localized on a cosmid clone by subcloning and Tn5::751 mutagenesis, was subjected to nucleotide sequencing. A single open reading frame, designated aad (amino acid deaminase), which appears to be both necessary and sufficient for deaminase activity, predicts a 473-amino-acid polypeptide (51,151 Da) encoded within an area mapped by transposon mutagenesis. The predicted amino acid sequence of Aad did not share significant amino acid sequence similarity with any other polypeptide in the PIR or SwissProt database. Amino acid deaminase activity in both P. mirabilis and E. coli transformed with aad-encoding plasmids was not affected by medium iron concentration or expression of genes in multicopy in fur, cya, or crp E. coli backgrounds. Enzyme expression was negatively affected by growth with glucose or glycerol as the sole carbon source but was not consistent with catabolite repression.     

Isolation, sequence, and expression in Escherichia coli of the Pseudomonas sp. strain ACP gene encoding 1-aminocyclopropane-1-carboxylate deaminase.

Pseudomonas sp. strain ACP is capable of growth on 1-aminocyclopropane-1-carboxylate (ACC) as a nitrogen source owing to induction of the enzyme ACC deaminase and the subsequent conversion of ACC to alpha-ketobutyrate and ammonia (M. Honma, Agric. Biol. Chem. 49:567-571, 1985). The complete amino acid sequence of purified ACC deaminase was determined, and the sequence information was used to clone the ACC deaminase gene from a 6-kb EcoRI fragment of Pseudomonas sp. strain ACP DNA. DNA sequence analysis of an EcoRI-PstI subclone demonstrated an open reading frame (ORF) encoding a polypeptide with a deduced amino acid sequence identical to the protein sequence determined chemically and a predicted molecular mass of 36,674 Da. The ORF also contained an additional 72 bp of upstream sequence not predicted by the amino acid sequence. Escherichia coli minicells containing the 6-kb clone expressed a major polypeptide of the size expected for ACC deaminase which was reactive with ACC deaminase antiserum. Furthermore, a lacZ fusion with the ACC deaminase ORF resulted in the expression of active enzyme in E. coli. ACC is a key intermediate in the biosynthesis of ethylene in plants, and the use of the ACC deaminase gene to manipulate this pathway is discussed.     

Participation of an extracellular deaminase in amino acid utilization by Neurospora crassa

A strain of Neurospora crassa defective in amino acid transport can utilize a variety of amino acids for growth when readily metabolizable nitrogen is limiting. Growth is accompanied by the production of an extracellular deaminase that converts the amino acid to its respective keto acid plus equimolar quantities of utilizable nitrogen in the ammonium ion form. Production of the deaminase is subject to ammonium repression. The relationship between the ability of an amino acid to trigger deaminase production and the presence of particular amino acid permease deficiencies is complex. Four classes of amino acids have been defined with respect to this relationship. The existence of multiple extracellular deaminases is discussed.     

Studies on ATP Deaminase

Water insoluble ATP deaminase was prepared by binding native ATP deaminase to DEAE-cellulose and several enzymatic properties of bound ATP deaminase were compared with those of free deaminase. The optimum pH of the former was 3.0 and shifted toward acid side by about 2 pH units compared with that of the latter. The activity of bound deaminase to ATP corresponded to about one fourth of free deaminase at pH 3.0. The possibility of continuous preparation of IMP from AMP by use of bound deaminase was suggested.     

过表达氨基葡萄糖脱氨酶对大肠杆菌氨基葡萄糖合成及中心碳代谢的影响

考察过表达氨基葡萄糖脱氨酶对氨基葡萄糖合成及大肠杆菌（Escherichia coli）中心碳代谢的影响。实验结果表明：过表达氨基葡萄糖脱氨酶使得在36 g/L葡萄糖,pH为9.0的发酵条件下,发酵24 h后,重组菌发酵液中氨基葡萄糖、丙酮酸和乙酸的量分别是对照菌Rosetta的2.1、1.48和1.74倍;而乳酸的量为2.53 g/L,对照菌Rosetta发酵液中的乳酸含量未检测到,重组菌发酵液中柠檬酸及α-酮戊二酸的含量分别是Rosetta的2.99和2.73倍。     

1-aminocyclopropane-1-carboxylate (ACC) deaminase induced by ACC synthesized and accumulated in Penicillium citrinum intracellular spaces

We have already described how 1-aminocyclopropane-1-carboxylic acid (ACC), which is a precursor of the plant hormone ethylene, is synthesized in Penicillium citrinum through the same reaction by the catalysis of ACC synthase [EC 4.4.1.14] as in higher plants. In addition, ACC deaminase [EC 4.1.99.4], which degrades ACC to 2-oxobutyrate and ammonia, was also purified from this strain. To study control of induction of ACC deaminase in this organism, we have isolated and analyzed the cDNA of P. citrinum ACC deaminase and studied the expression of ACC deaminase mRNA in P. citrinum cells. By the analysis of peptides from the digests of the purified and modified ACC deaminase with lysylendopeptidase, 70 % of its amino acid sequences were obtained. These amino acid sequences were used to identify a cDNA, consisting of 1,233 bp with an open reading frame of 1,080 bp encoding ACC deaminase with 360 amino acids. The deduced amino acids from the cDNA are identical by 52% and 45% to those of enzymes of Pseudomonas sp. ACP and Hansenula saturnus. Through Northern blot analysis, we found that the mRNA of ACC deaminase was expressed in P. citrinum cells grown in a medium containing 0.05% L-methionine. These findings suggest that ACC synthesized by ACC synthase and accumulated in P. citrinum intracellular spaces can induce the ACC deaminase that degrades the ACC.     

Regulation of chicken erythrocyte AMP deaminase by phytic acid.

AMP deaminase [EC 3.5.6.4] purified from chicken erythrocytes was inhibited by phytic acid (inositol hexaphosphate), which is the principal organic phosphate in chicken red cells. Kinetic analysis has indicated that this inhibition is of an allosteric type. The estimated Ki value was within the normal range of phytic acid concentration, suggesting that this compound acts as a physiological effector. Divalent cations such as Ca2+ and Mg2+ were shown to affect AMP deaminase by potentiating inhibition by lower concentrations of phytic acid, and by relieving the inhibition at higher concentrations of phytic acid. These results suggests that Ca2+ and Mg2+ can modify the inhibition of AMP deaminase by phytic acid in chicken red cells.     

Human adenosine deaminase. cDNA and complete primary amino acid sequence

A previously cloned partial adenosine deaminase cDNA insert (0.8 kilobase) was used to clone additional nucleotide sequences from human HPB ALL cDNA libraries. cDNA encompassing the entire coding, and 3'-untranslated regions as well as nearly all of the 5'-untranslated region was obtained. The complete amino acid sequence of the enzyme deduced from the cDNA sequence and protein sequencing consists of 362 amino acids, excluding the initiator Met, and accounts for Mr = 40,638. Secondary structure predictions assign adenosine deaminase to the alpha/beta class of proteins. Northern blot analysis with a cDNA probe showed adenosine deaminase mRNA to be present in normal to above normal amounts in B-lymphoblasts derived from adenosine deaminase-deficient patients with severe combined immunodeficiency disease. Knowledge of the cDNA and primary amino acid sequence of adenosine deaminase will be pivotal in further defining the genetic abnormality and its functional consequences in adenosine deaminase expression defects.     

Selenium as a novel regulator of porphyrin biosynthesis in germinating seedlings of mung bean (Phaseolus vulgaris)

5-Aminolevulinic acid, porphyrin and chlorophyll contents as well the activities of 5-aminolevulinic acid dehydratase and PBG deaminase were studied in selenium treated mung bean seedlings. Selenium had no effect on 5-aminolevulinic acid synthetic ability but inhibited 5-aminolevulinic acid dehydratase and PBG deaminase activities. Further, it was observed that selenium induced accumulation of protoporphyrin-IX and Mg-protoporphyrin ester and decreased chlorophyll levels in both light and dark-grown seedlings. The results suggest the possible regulatory role of selenium on chlorophyll synthesis by interacting with sulfhydryl containing enzymes 5-aminolevulinic acid dehydratase and porphobilinogen deaminase.     

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.     

Regulation of Expression of the prb-1b / ACC Deaminase Gene by UV-8 in Transgenic Tomatoes

Transgenic tomato plants with 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase gene from Enterobacter cloacae UW4 under the control of a pathogenesis-related promoter (prb-1b) from tobacco were challenged by abiotic stresses to determine the expression patterns ofthe transgene. No ACC deaminase RNA or protein was detected by RT-PCR and in western blots prepared from leaf proteins of transgenic plants after wounding or treatment with α-amino butyric acid, xylanase, ethephon, salicylic acid, jasmonic acid, ethylene, or ethylene plus jasmonic acid. However, expression of the ACC deaminase transgene was observed in leaves and roots oftransformed tomato lines exposed to UV light. The UV response required a minimum of 48 h of exposure and was specific to UV-8 light.     

Adenosine inhibits phenylephrine activation of phospholipase A in hamster brown adipocytes

Exposure of brown adipocytes to phenylephrine activates a phospholipase A2 producing arachidonic acid and lysophospholipids. When adipocytes were incubated with adenosine deaminase, a greater release of arachidonic acid and accumulation of lysophosphatidyl-choline in response to phenylephrine was noted. The potentiating effect of adenosine deaminase was also observed in the presence of A23187 and for both stimuli, the effect of adenosine deaminase was reversed by phenylisopropyladenosine. These results suggest the presence of an heretofore unrecognized action of adenosine, namely inhibition of phospholipase A2 activity in brown fat cells.     

Biosynthesis of Isoleucine and Valine in Rhodopseudomonas spheroides: Regulation of Threonine Deaminase Activity

The activities of threonine deaminase, acetohydroxy acid synthetase, acetohydroxy acid reductoisomerase, dihydroxy acid dehydrase, and transaminase B were detected in cell-free extracts of Rhodopseudomonas spheroides. No significant repression or derepression of threonine deaminase activity was observed.     

Simultaneous decrease in deamination of 5'-adenylic acid and 5'-deoxy-5'-S-isobutylthioadenosine in chick-embryo fibroblasts infected by Rous sarcoma virus

The rate of deamination of 5'-deoxy-5'-S-isobutylthioadenosine [(iBuS5'Ado] in chick embryo fibroblasts was substantially reduced after their infection and morphological transformation by Rous sarcoma virus. Concomitant with the reduction in rate of (iBuS)5'Ado deamination there was a decrease in adenosine deaminase and 5'-adenylic acid deaminase activities. The drop of these activities was related to infection and not to the expression of the src gene. (iBuS)5'Ado was deaminated by at least three enzymes or isoenzymes whose apparent molecular weights have been estimated to be 295000, 121000 and 37000 respectively. Two of these enzymes have been characterized as 5'-adenylic acid deaminase and the heavy form of adenosine deaminase, respectively.     

Folic acid and pterin deaminases in Dictyostelium discoideum: kinetic properties and regulation by folic acid, pterin, and adenosine 3'',5''-phosphate.

Kinetic data obtained for deamination of pterin by the extracellular fraction from Dictyostelium discoideum yielded apparently linear Lineweaver-Burk plots for pterin. The Michaelis constant for pterin was 30 microM. The data for folic acid deamination yielded convex Lineweaver-Burk plots. Convex Lineweaver-Burk plots could result from the presence of two types of enzymes with different affinities. The data for folic acid deamination were analyzed mathematically for two types of enzymes. This analysis produced Michaelis constants for folic acid of 1.8 and 23 microM competition studies suggested that an enzyme with low affinity nonspecifically catalyzed the deamination of folic acid and pterin, whereas an enzyme with high affinity was a specific folic acid deaminase. A specific folic acid deaminase with high affinity appeared to be present on the surface of D. discoideum cells. The Michaelis constant for this enzyme was 2.6 microM. Cells growing in nutrient broth and cells starved in phosphate buffer released folic acid and pterin deaminases. The quantity of deaminase activities released by the cells appeared to be controlled by chemoattractants. Starving cells that were supplied with folic acid, pterin, or adenosine 3',5'-phosphate increased their extracellular folic acid and pterin deaminase activities to a larger extent than did cell suspensions to which no chemoattractants were added. Administration of folic acid or pterin to starving cells caused increases of the activity of extracellular adenosine 3',5'-phosphate phosphodiesterase and repressed increases of the activity of phosphodiesterase inhibitor.     

Folic acid deaminase activity during development in Dictyostelium discoideum.

Folic acid attracts vegetative amoebae of Dictyostelium discoideum. Secreted by bacteria, it may act as a food-seeking device. The inactivation of this attractant is catalyzed by a deaminase. As assay has been developed to measure the folic acid deaminase activity. In addition to cell-surface an intracellular deaminase, the amoebae of D. discoideum release the enzyme into the medium. The pH optimum of the extracellular enzyme was 6.0, and higher for the cell-associated deaminases. The extracellular enzyme was secreted maximally by vegetative amoebae, and its activity diminished during cell differentiation. The cell-surface bound enzyme was less active than the extracellular enzyme, and its activity decreased twofold during a 6-h starvation period. The enzyme activity of homogenates and 48,000 x g pellets diminished during this period 35 to 40%. The supernatant of a homogenate had a higher deaminase activity than the homogenate itself or its pellet; this suggests the presence of an inhibitor in the particulate fraction. The underlying mechanism for inactivation of folic acid has similar characteristics as that for inactivation of cyclic adenosine monophosphate.     

The regulatory role of spermine and fatty acid in the interaction of AMP deaminase with phosphofructokinase

The role of fatty acid and polyamine in the interaction of AMP deaminase (EC 3.5.4.6)-ammonium system with glycolysis was investigated using permeabilized yeast cells. (1) The addition of fatty acid inhibited the activity of AMP deaminase in situ, resulting in a decrease in the total adenylate pool depletion, and in the recovery of the adenylate energy charge. (2) The addition of fatty acid resulted in an indirect decrease in the activity of phosphofructokinase (EC 2.7.1.11) through a reduced level of ammonium ion; fatty acid itself did not inhibit phosphofructokinase activity in the presence of excess ammonium ion. (3) Spermine protected AMP deaminase from inhibition by fatty acid: the increased ammonium level enhanced phosphofructokinase activity, glycolytic flux and the recovery of the energy charge. In contrast, alkali metals, which are also activators of AMP deaminase had little effect on the inhibition of the enzyme. The inhibition of glycolysis by fatty acid and its reversal by polyamine can be accounted for by the changes in ammonium ion through the action of AMP deaminase-ammonium system, and the physiological relevance is discussed.     

ECTO-enzyme activity of human erythrocyte adenosine deaminase

Summary Adenosine deaminase is found primarily in the cytoplasm of many cell types. In the human erythrocyte, about 30 per cent of the total adenosine deaminase activity is membrane associated, and about two-thirds of this is inactivated by treatment of intact erythrocytes with the nonpenetrating reagent diazotized sulfanilic acid, without affecting lactate dehydrogenase, a soluble cytoplasmic enzyme. This indicates that within the cell membranes, the catalytic site of about two-thirds of the adenosine deaminase faces the external medium, i.e., ecto adenosine deaminase. Localization of adenosine deaminase activity at the cell membrane is demonstrated directly by electron microscopy by use of the substrate 6-Chloropurine ribonucleoside, which is dechlorinated by adenosine deaminase to produce Cl^–, which is precipitated at its locus of formation by added Ag⁺, and the precipitated AgCl converted into the electron dense Ag⁰ upon exposure to light.From the Hydropathic Profile of the amino acid sequence of adenosine deaminase it is evident that there are two hydrophobic domains of sufficient length to span a biological membrane, and it is proposed that these domains could function to anchor the enzyme to the membrane.The importance of adenosine deaminase is indicated by the fatal immuno-deficiency which results from untreated genetic adenosine deaminase deficiency. It may be important to determine whether the amount of ecto adenosine deaminase activity is better suited to assess the clinical status of adenosine deaminase deficient patients that the currently used total cellular enzyme activity.Abbreviations ADA Adenosine Deaminase - LDH Lactate Dehydrogenase - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid - CPR 6-Chloropurine Ribonucleoside - SDS Sodium Dodecyl Sulfate - NAD -Nicotinamide Adenine Dinucleotide - HBSS Hank's Balanced Salt Solution - DASA Diazotized Sulfanilic Acid     

Role for Free Isoleucine or Glycyl-Leucine in the Repression of Threonine Deaminase in Escherichia coli

In Escherichia coli, the three branched-chain amino acid activating enzymes appear to be essential for multivalent repression of the isoleucine- and valine-forming enzymes. The results of experiments with a mutant, strain CU18, having an altered threonine deaminase, indicate that free isoleucine and some form of threonine deaminase (the product of the ilvA gene) are also involved in multivalent repression. This strain exhibits abnormally high derepressibility but normal repressibility of its ilv gene products, and its threonine deaminase is inhibited only by high concentrations of isoleucine. In strain CU18, the isoleucine analogue, thiaisoleucine, is incapable of replacing isoleucine in the multivalent repression of the ilv genes, whereas the analogue can fully replace the natural amino acid in repression in other strains examined. The dipeptide, glycyl-leucine, which, like isoleucine, is a heterotropic negative effector of threonine deaminase but is not a substrate for isoleucyl-transfer ribonucleic acid synthetase, can completely prevent the accumulation of threonine deaminase-forming potential during isoleucine starvation in strains with normal threonine deaminases. It can not, however, prevent such accumulation in strain CU18 or in other strains in which threonine deaminase is insensitive to any concentration of isoleucine.