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Query: UNIPROT:P06889 (Mol)
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Rhizobium meliloti strain GRM8 is able to transform ornithine into proline by means of an ornithine cyclodeaminase and, therefore, has the ability to use either of these amino acids as its sole carbon and nitrogen source. By Tn5 insertion mutagenesis we obtained a GRM8 mutant derivative strain (LM1) unable to catabolize either ornithine or proline. DNA hybridization studies showed that the LM1 mutant carries a single Tn5 insertion within a chromosomally located gene that, as deduced from a partial nucleotide sequence, encodes a proline dehydrogenase (ProDH). Enzymatic assays confirmed the lack of ProDH activity in cell extracts of strain LM1 and revealed that production of this enzyme is inducible in the parental strain by proline and ornithine. Ultrastructural nodule microscopy analysis, acetylene reduction assays, and dry-weight determinations of nodulated alfalfa plants showed no obvious defect in the nitrogen fixation process of the ProDH- mutant LM1. However, nodulation tests and competition assays demonstrated that in R. meliloti ProDH is required for nodulation efficiency and competitiveness on alfalfa roots.
Mol Plant Microbe Interact
PMID:Characterization of a Rhizobium meliloti proline dehydrogenase mutant altered in nodulation efficiency and competitiveness on alfalfa roots. 858 6

Helix formation in a 17-residue alanine-lysine peptide and analogous peptides with specific lysine --> X substitutions, where X is 2,3-diamino-L-propionic acid, 2, 4-diamino-L-butyric acid or L-ornithine, have been examined using circular dichroism measurements. The dependence of helix content on X, its position in the sequence, and the number of lysine --> X substitutions are reasonably well described by using the Lifson-Roig theory modified to include N-capping, without explicitly considering charge-helix dipole interactions. The helix propensities for these basic amino acids increase with the length of the side-chain in the rank order 2,3-diamino-L-propionic acid < 2,4-diamino-L-butyric acid < ornithine < lysine. This parallels the increase in helix propensities with side-chain length of other polar and charged amino acids.
J Mol Biol 1996 Apr 05
PMID:Helix propensities of basic amino acids increase with the length of the side-chain. 864 36

Copper diamine oxidase from lentil (Lens culinaris) seedlings was shown to be able to catalyze the oxidative deamination of a wide range of aliphatic and aromatic monoamine compounds, including some amino acids. Although the catalytic efficiencies were only 1-3% of that measured with the diamine substrate putrescine, they were still comparable to those of specialized monoamine oxidases. In particular, the lentil enzyme oxidized benzylamine and histamine with K(m) and Vmax values similar to those found for the mammalian enzymes benzylamine oxidase and histaminase. Cysteamine was found to be a substrate of the enzyme, whereas hypotaurine and taurine were found to be neither substrates nor inhibitors of the enzyme. Quite unexpectedly the amino acids L-ornithine and L-lysine were oxidized by lentil enzyme, and beta-alanine and gamma-aminobutyric acid were oxidized only at high concentrations of enzyme. These results suggest that enzymes normally classified as diamine oxidases could in fact have a more diversified role in metabolism than recognized so far.
Biochem Mol Biol Int 1996 Oct
PMID:Substrate specificity of lentil seedling amine oxidase. 890 74

We used a PCR-based library screening method to isolate a 1.4 kb pea leaf cDNA encoding ornithine transcarbamoylase (OTCase). The cDNA contains a single major ORF of 375 amino acids whose deduced sequence exhibits a high degree of homology with other OTCases. The predicted molecular mass of 41361 Da for this protein is approximately the 40 kDa size of the polypeptide that is immunoprecipitated with OTCase antibody after in vitro translation of pea leaf mRNA. In vivo, OTCase occurs as a trimer of identical 36.5 kDa polypeptides, suggesting that this enzyme is synthesized as a cytosolic precursor protein. Southern blot analysis indicates that multiple OTCase genes occur in pea. An abundant 1.4 kb transcript is seen in northern blots of total RNA isolated from the leaves and roots of light- and dark-grown pea seedlings.
Plant Mol Biol 1996 Sep
PMID:Isolation and characterization of a cDNA encoding a pea ornithine transcarbamoylase (argF) and comparison with other transcarbamoylases. 891 25

Positively charged cyclic peptides (three to seven amino acids) have been tested for their inhibitory effects on Na+/Ca2+ exchange in the cardiac sarcolemma vesicles. The lead structure of Phe-Arg-Cys-Arg-Cys-Phe-CONH2 (FRCRCFa) has been systematically modified for identification of important pharmacophores. In cyclic peptides (intramolecular S-S bond, the carboxyl terminal is locked with amide (CONH2), and positive charge is retained by one or two arginines, ornithines, or lysines. Thirty-five different cyclic peptides show IC50 values in the range of 2-800 microM, suggesting that some specific structure-activity relationships may determine the inhibitory effects. Shortening of the FRCRCFa length to four amino acids decreases the inhibitory potency by 10-80-fold. The substitution of Arg2 or Arg4 in FRCRCFa with lysine or ornithine decreases the inhibitory potency by 5-12-fold, suggesting that both arginines are beneficial for inhibition. The substitution of Phe1 in FRCRCFa by 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid produces a potent inhibitor (IC50 = 2-4 microM). The N-myristoylated FRCRCFa exhibits an inhibitory potency (IC50 = 8-10 microM) similar to that of the parent FRCRCFa peptide, thereby arousing a new possibility for the development of a cell-permeable blocker of the Na+/Ca2+ exchanger, D-Arg4 or D-Cys5 substitutions in FRCRCFa do not alter the inhibitory effect, whereas the L-to-D substitutions of other amino acids in FRCRCFa reduce the inhibitory potency by 4-5-fold. Thus, the L-to-D substitutions of Arg4 and/or Cys5 have a potential to increase the peptide stability to proteolytic degradation. The insertion of proline outside of the ring of FRCRCFa diminishes the inhibitory potency by 3-6-fold, whereas proline introduction into the ring decreases the inhibitory potency by 16-20-fold. The replacement of Cys3 and Cys5 in FRCRCFa with beta, beta-dimethylcystein has no significant effect on the inhibitory potency, suggesting that the S-S bond is not exposed to the interface of the peptide/receptor interaction. In conclusion, the current data support a proposal that the conformationally constrained Arg-Cys-Arg-Cys structure is obligatory for inhibition of Na+/Ca2+ exchange, whereas hydrophobic additions at the carboxyl and amino ends have limited effects in increasing the inhibitory potency.
Mol Pharmacol 1997 Jan
PMID:Inhibition of the cardiac sarcolemma Na+/Ca2+ exchanger by conformationally constrained small cyclic peptides. 901 54

We describe the molecular and functional characterization of three closely related S-adenosyl-L-methionine synthetase (SAMS) isoenzymes from Catharanthus roseus (Madagascar periwinkle). The genes are differentially expressed in cell cultures during growth of the culture and after application of various stresses (elicitor, nutritional down-shift, increased NaCl). Seedlings revealed organ-specific expression and differential gene regulation after salt stress. A relationship analysis indicated that plant SAMS group in two main clusters distinguished by characteristic amino acid exchanges at specific positions, and this suggested differences in the enzyme properties or the regulation. SAMS1 and SAMS2 are of type I and SAMS3 is of type II. The properties of the isoenzymes were compared after heterologous expression of the individual enzymes, but no significant differences were detected in a) optima for temperature (37 to 45 degrees C) or pH (7 to 8.3); b) dependence on cations (divalent: Mg2+, Mn2+, Co2+; monovalent: K+, NH4+, Na+); c) K(m)s for ATP and L-methionine; d) inhibition by reaction products (S-adenosyl-L-methionine, PPi, Pi), by the reaction intermediate tripolyphosphate, and by the substrate analogues ethionine and cycloleucine; e) response to metabolites from the methyl cycle (L-homocysteine) or from related pathways (L-ornithine, putrescine, spermidine, spermine); f) native protein size (gel permeation chromatography). The results represent the first characterization of plant SAMS isoenzyme properties with individually expressed proteins. The possibility is discussed that the isoenzyme differences reflect specificities in the association with enzymes that use S-adenosyl-L-methionine.
Plant Mol Biol 1997 Jan
PMID:Three differentially expressed S-adenosylmethionine synthetases from Catharanthus roseus: molecular and functional characterization. 903 40

Polyamines play an important and central role in normal cell growth and differentiation in many cells. In trypanosomatids, spermidine is also an essential precursor in the biosynthesis of the unique glutathione-spermidine conjugate, trypanothione. Our previous study has shown that the epimastigote stage of Trypanosoma cruzi (Silvio strain) is incapable of significant de novo synthesis of putrescine or cadaverine from their amino acid precursors [Hunter, Le Quesne and Fairlamb (1994) Eur. J. Biochem. 226, 1019-1027]. In this study we show that when grown to late log phase in medium containing trace amounts of putrescine (0.22 microM) and spermidine (0.63 microM), Y-strain epimastigotes contain low levels of polyamines with free glutathione as their principal low molecular mass thiol (> 97% of total glutathione). Following passage into fresh medium, trypanothione and glutathionylspermidine content increase to 46% of total glutathione by mid log phase but returns to less than 3% by late log phase. In contrast, when supplemented at inoculation with exogenous putrescine, glutathione-spermidine conjugates reach 80% of total glutathione by early log phase and remain elevated throughout growth. Supplementation with exogenous putrescine or spermidine during polyamine starvation (late log phase) results in increased conjugate levels (> 74% of total glutathione) and is associated with large increases in total putrescine and spermidine. Likewise, supplementation with exogenous cadaverine and aminopropylcadaverine results in similar increases in trypanothione analogues and total cadaverine and aminopropylcadaverine. In contrast, ornithine, arginine, lysine, agmatine and other amino acid precursors have no effect on polyamine or conjugate levels. No significant ornithine or arginine decarboxylase activities could be detected (< 0.8 pmol min-1 [mg protein]-1). Similar results were obtained for epimastigotes representing all the major zymodeme classes, providing evidence that diamine auxotrophy may be a universal feature of this stage of the life-cycle.
Mol Biochem Parasitol 1997 Jan
PMID:Diamine auxotrophy may be a universal feature of Trypanosoma cruzi epimastigotes. 904 26

The presence of a tumour significantly changes nitrogen metabolism, including that of amino acids and polyamines, in host animals. In this study, we examine whether developing tumours affect the metabolic relationship of arginine and ornithine, precursors of polyamines, in the testosterone-induced hypertrophic mouse kidney model. Androgen-induced changes in the activity of enzymes involved with ornithine biosynthesis (arginase), its consumption (ornithine aminotransferase, OAT and ornithine decarboxylase, ODC) and the hypertrophy of host mouse kidney were not affected by the presence of an ascitic tumour (EAC) and only slightly by a mammary carcinoma (MaCa). The HPLC determined renal level of arginine and ornithine showed a striking homeostasis and was disturbed neither by testosterone nor EAC. The effect of MaCa and testosterone on the levels of both amino acids, although significant, was not very pronounced. Developing tumours, especially ascitic, altered the renal activity of OAT and ODC, but not of arginase, in testosterone-untreated mice. All examined tumours, EAC, L 1210 and MaCa actively metabolized arginine and ornithine. the tumour content of arginine which coincided with the activity of arginase, resulted in a marked increase of the ornithine/arginine ratio in tumours, when compared with kidneys. These results indicate that the androgen-induced anabolic response in mouse kidney is preserved, in spite of tumour requirements for essential metabolites.
Mol Cell Biochem 1997 Mar
PMID:Tumour effect on arginine/ornithine metabolic relationship in hypertrophic mouse kidney. 906 93

A method for modeling large-scale rearrangements of protein domains connected by a single- or a double-stranded linker is proposed. Multidomain proteins may undergo substantial domain displacements, while their intradomain structure remains essentially unchanged. The method allows automatic identification of an interdomain linker and builds an all-atom model of a protein structure in internal coordinates. Torsion angles belonging to the interdomain linkers and side chains potentially able to form domain interfaces are set free while remaining torsions, bond lengths, and bond angles are fixed. Large-scale sampling of the reduced torsions conformational subspace is effected with the "biased probability Monte Carlo-minimization" method [Abagyan, R.A., Totrov, M.M. (1994): J. Mol. Biol. 235, 983-1002]. Solvation and side-chain entropic contributions are added to the energy function. A special procedure has been developed to generate concerted deformations of a double-stranded interdomain linker in such a way that the polypeptide chain continuity is preserved. The method was tested on Bence-Jones protein with a single-stranded linker and lysine/arginine/ornithine-binding (LAO) protein with a double-stranded linker. For each protein, structurally diverse low-energy conformations with ideal covalent geometry were generated, and an overlap between two sets of conformations generated starting from the crystallographically determined "closed" and "open" forms was found. One of the low-energy conformations generated in a run starting from the LAO "closed" form was only 2.2 A away from the structure of the "open" form. The method can be useful in predicting the scope of possible domain rearrangements of a multidomain protein.
 ...
PMID:A new method for modeling large-scale rearrangements of protein domains. 909 43

As a toxic metabolic byproduct in mammals, excess ammonia is converted into urea by a series of five enzymatic reactions in the liver that constitute the urea cycle. A portion of this cycle takes place in the mitochondria, while the remainder is cytosolic. Liver arginase (L-arginine ureahydrolase, A1) is the fifth enzyme of the cycle, catalyzing the hydrolysis of arginine to ornithine and urea within the cytosol. Patients deficient in this enzyme exhibit hyperargininemia with episodic hyperammonemia and long-term effects of mental retardation and spasticity. However, the hyperammonemic effects are not so catastrophic in arginase deficiency as compared to other urea cycle defects. Earlier studies have suggested that this is due to the mitigating effect of a second isozyme of arginase (AII) expressed predominantly in the kidney and localized within the mitochondria. In order to explore the curious dual evolution of these two isozymes, and the ways in which the intriguing, aspects of AII physiology might be exploited for gene replacement therapy of AI deficiency, the cloned cDNA for human AI was inserted into an expression vector downstream from the mitochondrial targeting leader sequence for the mitochondrial enzyme ornithine transcarbamylase and transfected into a variety of recipient cell types. AI expression in the target cells was confirmed by northern blot analysis, and competition and immunoprecipitation studies showed successful translocation of the exogenous AI enzyme into the transfected cell mitochondria. Stability studies demonstrated that the translocated enzyme had a longer half-life than either native cytosolic AI or mitochondrial AII. Incubation of the transfected cells with increasing amounts of arginine produced enhanced levels of mitochondrial AI activity, a substrate-induced effect that we have previously seen with native AII but never AI. Along with exploring the basic biological questions of regulation and subcellular localization in this unique dual-enzyme system, these results suggest that the mitochondrial matrix space may be a preferred site for delivery of enzymes in gene replacement therapy.
Somat Cell Mol Genet 1996 Nov
PMID:Delivery of cytosolic liver arginase into the mitochondrial matrix space: a possible novel site for gene replacement therapy. 913 Oct 18


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