Allosteric regulation of serine hydroxymethyltransferase from mung bean (Phaseolusaureus)

Serine hydroxymethyltransferase, the first enzyme in the pathway for the interconversion of one carbon compounds was purified from mung bean seedlings by ammonium sulfate fractionation, DEAE-Sephadex, Blue Sepharose CL-6B affinity chromatography and gel filteration on Sephacryl S-200. The specific activity of the enzyme, 0.73 (u mol HCHO formed/min/mg protein) was 10⁴ times larger than the highest value reported hitherto. Saturation of tetrahydrofolate was sigmoid, whereas with serine was hyperbolic, with n_H values of 1.9 and 1.0 respectively. Reduced nicotinamide adenine dinucleotide, lysine and methionine decreased, whereas nicotinamide adenine dinucleotide, adenosine 5′-monophosphate and adenosine 5′-triphosphate increased the sigmoidicity. These results suggest that serine hydroxymethyltransferase from mung bean is a regulatory enzyme.     

Purification and Characterization of the Pseudomonas multivorans Glucose-6-Phosphate Dehydrogenase Active with Nicotinamide Adenine Dinucleotide

The Pseudomonas multivorans glucose-6-phosphate dehydrogenase (EC 1.1.1.49) active with nicotinamide adenine dinucleotide, which is inhibitable by adenosine-5'-triphosphate, was purified approximately 1,000-fold from extracts of glucose-grown bacteria, and characterized with respect to subunit composition, response to different inhibitory ligands, and certain other properties. The enzyme was found to be an oligomer composed of four subunits of about 60,000 molecular weight. Reduced nicotinamide adenine dinucleotide phosphate, but not reduced nicotinamide adenine dinucleotide, was found to be a potent inhibitor of its activity. The range of concentrations of reduced nicotinamide adenine dinucleotide phosphate over which inhibition occurred was about 100-fold lower than that for adenosine-5'-triphosphate. The data suggest that reduced nicotinamide adenine dinucleotide phosphate may play an important role in regulation of hexose phosphate metabolism in P. multivorans. Antisera prepared against the purified enzyme strongly inhibited its activity, but failed to inhibit the activity of the nicotinamide adenine dinucleotide phosphate-specific glucose-6-phosphate dehydrogenase which is also present in extracts of this bacterium. Immunodiffusion experiments confirmed the results of the enzyme inhibition studies, and failed to support the idea that the two glucose-6-phosphate dehydrogenase species from P. multivorans represent different oligomeric forms of the same protein.     

Further chemical characterization and biological activities of fluorescent I,N6-ethenoadenosine derivatives

The fluorescent 1,N⁶-ethenoadenosine derivatives of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, 3′:5′-cyclic adenosine monophosphate, adenosine and nicotinamide adenine dinucleotide have been prepared. Paper and thin layer chromatographic purification methods have been developed. Nuclear magnetic resonance and mass spectrum data indicate that only the purine ring has been modified.The 1,N⁶-ethenoadenosine triphosphate had about 70% of the activity of adenosine triphosphate as a substrate for total adenosine triphosphatase activity of hypophysectomized rat liver membranes. The 1,N⁶-ethenoadenosine diphosphate had about 86% of the activity of adenosine diphosphate as a substrate for adenosine diphosphatase of hypophysectomized rat liver membranes. The 1,N⁶-etheno derivative of nicotinamide adenine dinucleotide had about 8% of the activity of nicotinamide adenine dinucleotide as a substrate for nicotinamide adenine dinucleotide glycohydrolase and about 54% of the activity of nicotinamide adenine dinucleotide as a substrate for nicotinamide adenine dinucleotide pyrophosphatase of hypophysectomized rat liver membranes.K_m's for the ATPase, ADPase and yeast alcohol dehydrogenase using ε-ATP and ε-ADP and ε-NAD as substrates are presented.     

Purification and some properties of carbon monoxide dehydrogenase from Pseudomonas carboxydohydrogena

A soluble yellow CO dehydrogenase from CO-autotrophically grown cells of Pseudomonas carboxydohydrogena was purified 35-fold in seven steps to better than 95% homogeneity with a yield of 30%. The final specific activity was 180 μmol of acceptor reduced per min per mg of protein as determined by an assay based on the CO-dependent reduction of thionin. Methyl viologen, nicotinamide adenine dinucleotide (phosphate), flavin mononucleotide, and flavin adenine dinucleotide were not reduced by the enzyme, but methylene blue, thionin, and toluylene blue were reduced. The molecular weight of native enzyme was determined to be 4 × 10⁵. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed at least three nonidentical subunits of molecular weights 14,000 (α), 28,000 (β), and 85,000 (γ). The ratio of densities of each subunit after electrophoresis was about 1:2:6 (α/β/γ), suggesting an α₃β₃γ₃ structure for the enzyme. The purified enzyme was free of formate dehydrogenase and nicotinamide adenine dinucleotide-specific hydrogenase activities, but contained particulate hydrogenase-like activity with thionin as electron acceptor. Known metalchelating agents tested had no effect on CO dehydrogenase activity. No divalent cations tested stimulated enzyme activity. The native enzyme does not contain Ni since cells assimilated little ⁶³Ni during growth, and the specific ⁶³Ni content of the enzyme declined during purification. The isoelectric point of the native enzyme was found to be 4.5 to 4.7. The K_m for CO was found to be 63 μM. The spectrum of the enzyme and its protein-free extract revealed that it contains bound flavin. The cofactor was flavin adenine dinucleotide based on enzyme digestion and thin-layer chromatography. One mole of native enzyme contains at least 3 mol of noncovalently bound flavin adenine dinucleotide.     

A new spin-labelled analogue of nicotinamide adenine dinucleotide

The preparation of a spin-labelled analogue of nicotinamide adenine dinucleotide, 3-(4′,4′,5′,5′-tetramethyl-3′-oxide-1′-oxyl-2′-imidazolinyl) pyridine adenine dinucleotide, is descrebed. This compound was obtained by treatment of 3-carboxaldehyde pyridine adenine dinucleotide with 2,3-dimethyl-2,3-dihydroxylaminobutane followed by oxidation with lead dioxide. The interpretation of the particular electron spin resonance spectra of this nitronylnitroxide (five lines) in terms of the rotational correlation time of the radical is shown to be possible. The high stability of this compound makes its use in NAD⁺-dependent biological systems feasible.     

Nicotinamide Adenine Dinucleotide Phosphate-specific Isocitrate Dehydrogenase from a Higher Plant: Isolation and Characterization 1

Nicotinamide adenine dinucleotide phosphate-specific isocitrate dehydrogenase was extracted from etiolated pea (Pisum sativum L.) seedlings and was purified 65-fold. The purified enzyme exhibits one predominant protein band by polyacrylamide gel electrophoresis, which corresponds to the dehydrogenase activity as measured by the nitro blue tetrazolium technique. The reaction is readily reversible, the pH optima for the forward (nicotinamide adenine dinucleotide phosphate reduction) and reverse reactions being 8.4 and 6.0, respectively. The enzyme has different cofactor and inhibitor characteristics in the two directions. Manganese ions can be used as a cofactor for the reaction in each direction but magnesium ions only act as a cofactor in the forward reaction. Zinc ions, and to a lesser extent calcium ions, inhibit the enzyme at low concentrations when magnesium but not manganese is the metal activator. It is suggested that there is a fundamental difference between magnesium and manganese in the activation of the enzyme. The enzyme shows normal kinetics and the Michaelis contant for each substrate was determined. The inhibition by nucleotides, nucleosides, reaction products, and related compounds was studied. The enzyme shows a linear response to the mole fraction of reduced nicotinamide adenine dinucleotide phosphate when total nicotinamide adenine dinucleotide phosphate (nicotinamide adenine dinucleotide phosphate plus reduced nicotinamide adenine dinucleotide phosphate) is kept constant. Isocitrate in the presence of divalent metal ions will protect the enzyme from inactivation by p-chloromercuribenzoate. Protection is also afforded by manganese ions alone but not by magnesium ions alone There is a concerted inhibition of the enzyme by oxalacetate and glyoxylate.     

In-gel activity staining of oxidized nicotinamide adenine dinucleotide kinase by blue native polyacrylamide gel electrophoresis

Oxidized nicotinamide adenine dinucleotide (NAD(+)) kinase (NADK, E.C. 2.7.1.23) plays an instrumental role in cellular metabolism. Here we report on a blue native polyacrylamide gel electrophoretic technique that allows the facile detection of this enzyme. The product, oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)), formed following the reaction of NADK with NAD(+) and adenosine 5'-triphosphate was detected with the aid of glucose-6-phosphate dehydrogenase or NADP(+)-isocitrate dehydrogenase, iodonitrotetrazolium chloride, and phenazine methosulfate. The bands at the respective activity sites were excised and subjected to native and denaturing two-dimensional electrophoresis for the determination of protein levels. Hence this novel electrophoretic method allows the easy detection of NADK, a critical enzyme involved in pyridine homeostasis. Furthermore, this technique allowed the monitoring of the activity and expression of this kinase in various biological systems.     

Role of molybdenum in nitrate reduction by chlorella

Molybdenum is absolutely required for the nitrate-reducing activity of the nicotinamide adenine dinucleotide nitrate reductase complex isolated from Chlorella fusca. The whole enzyme nicotinamide adenine dinucleotide nitrate reductase is formed by cells grown in the absence of added molybdate, but only its first activity (nicotinamide adenine dinucleotide diaphorase) is functional. The second activity of the complex, which subsequently participates also in the enzymatic transfer of electrons from nicotinamide adenine dinucleotide to nitrate (FNH₂-nitrate reductase), depends on the presence of molybdenum. Neither molybdate nor nitrate is required for nitrate reductase synthesis de novo, but ammonia acts as a nutritional repressor of the complete enzyme complex. Under conditions which exclude de novo synthesis of nitrate reductase, the addition of molybdate to molybdenum-deficient cells clearly increases the activity level of this enzyme, thus suggesting in vivo incorporation of the trace metal into the pre-existing inactive apoenzyme.     

Catabolism of Phloroglucinol by the Rumen Anaerobe Coprococcus

A rumen isolate, Coprococcus, sp. Pe₁5, was found to carry phloroglucinol reductase, which catalyzed the initial step in the breakdown of phloroglucinol. The organism uses phloroglucinol as the sole source of carbon and energy when grown in the absence of oxygen. Induced levels of enzyme were detected in cells grown either on phloroglucinol or on other carbon sources in the presence of limiting quantities of phloroglucinol. Although the organism is a strict anaerobe, the enzyme from anaerobically grown cells was insensitive to air. The partially purified enzyme required reduced nicotinamide adenine dinucleotide phosphate as an electron donor and was specific for phloroglucinol. However, partial enzyme activity (14 to 17%) was also detected in the presence of 2-methyl-1,4-naphthoquinone but not in the presence of several other phenolic compounds. The enzyme exhibited a higher affinity for phloroglucinol than for reduced nicotinamide adenine dinucleotide phosphate, with K_m values of 3.0 × 10⁻⁵ M and 29.0 × 10⁻⁵ M, respectively. The optimum pH for maximal enzyme activity was 7.4, and the molecular weight of the native protein was about 130,000, as determined by the Sephadex gel filtration technique.     

Modification of Human Erythrocyte Pyruvate Kinase by an Active Site-directed Reagent: Bromopyruvate

Human erythrocyte pyruvate kinase was modified with bromopyruvate and the kinetic behavior of the modified enzyme was investigated. When the enzyme was modified with bromopyruvate in the absence of adenosine-5′s-diphosphate, phospho-enolpyruvate or fructose-1,6-diphosphate the inactivation followed a pseudo first-order kinetics. The inactivation rate constant, k_s, was 1.84 × 0.15 min^?1. K_d of the bromopyruvate-enzyme complex was 0.14 × 0.03 mM.

The presence of adenosine-5′-diphosphate, phosphoenolpyruvate or fructose-1,6-diphosphate in the modification medium or the presence of fructose-1,6-diphosphate in the assay medium resulted in deviation of the inactivation kinetics from pseudo first-order. Phosphoenolpyruvate was better than adenosine-5′-diphosphate for protection against bromopyruvate modification whereas fructose-1,6-diphosphate was ineffective. The modified enzyme showed negative cooperativity in the presence of fructose-1,6-diphosphate whereas in the absence of it no activity was detected.     

Alanine dehydrogenase of the N2-fixing blue-green alga,Anabaena cylindrica

The l-alanine dehydrogenase (ADH) of Anabaena cylindrica has been purified 700-fold. It has a molecular weight of approximately 270000, has 6 sub-units, each of molecular weight approximately 43000, and shows activity both in the aminating and deaminating directions. The enzyme is NADH/NAD⁺ specific and oxaloacetate can partially substitute for pyruvate. The K _m ^app for NAD⁺ is 14 M and 60 M at low and high NAD⁺ concentrations, respectively. The K _m ^app for l-alanine is 0.4 mM, that for pyruvate is 0.11 mM, and that for oxaloacetate is 3.0 mM. The K _m ^app for NH ₄ ⁺ varies from 8–133 mM depending on the pH, being lowest at high pH levels (pH 8.7 or above). Alanine, serine and glycine inhibit ADH activity in the aminating direction. The enzyme is active both in heterocysts and vegetative cells and activity is higher in nitrogen-starved cultures than in N₂-fixing cultures. The data suggest that although alanine is formed by the aminating activity of ADH, entry of newly fixed ammonia into organic combination does not occur primarily via ADH in N₂-fixing cultures of A. cylindrica. Ammonia assimilation via ADH may be important in cultures with an excess of available nitrogen. The deaminating activity of the enzyme may be important under conditions of nitrogen-deficiency.Abbreviations ADH alanine dehydrogenase - DEAE diethylamino ethyl cellulose - EDTA ethylenediamine tetraacetic acid - GDH glutamic dehydrogenase - GS glutamine synthetase - GOT aspartate-glutamate aminotransferase - NAD⁺ nicotinamide adenine dinucleotide - NADH reduced nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - NADPH reduced nicotinamide adenine dinucleotide phosphate - SDS sodium dodecyl sulphate - Tris tris(hydroxymethyl) aminomethane     

A new ternary complex between horse liver alcohol dehydrogenase, NAD+, and acetone

Acetone was found to form a dead-end ternary complex with horse liver alcohol dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD⁺) when the reactants were incubated for a long time at relatively high concentrations. The complex formation was demonstrated by measuring the increase in absorbance at 320 nm, the quenching of protein fluorescence, and the loss of enzyme activity. Since acetone is a substrate of liver alcohol dehydrogenase, and the presence of acetaldehyde or pyrazole prevents acetone from forming the dead-end complex with liver alcohol dehydrogenase and NAD⁺, the acetone molecule in the complex may be bound to the substrate binding site of liver alcohol dehydrogenase. The dissociation of the complex was demonstrated by prolonged dialysis or by addition of reduced nicotinamide adenine dinucleotide (NADH) and iso-butyramide. A modified nicotinamide adenine dinucleotide was obtained as a main product from the dead-end complex after dissociation of the complex or denaturation of the apoenzyme. The modified nicotinamide adenine dinucleotide was found to exhibit an absorption spectrum similar to that of NADH; however, it was not oxidizable by liver alcohol dehydrogenase in the presence of acetaldehyde and exhibited no fluorescence.     

Studies on Metabolic Pathway of NAD in Yeast

The properties of yeast 5′-nucleotidase, one of NAD-metabolic system in yeast, were studied.

1) The enzyme has optimum pH at 5.8~6.1 for its activity and is most stable at pH 6. It is inactivated completely at 55°C for 6 min, pH 7, but never at 40°C for 6 min. 2) The enzyme hydrolyzes only 5′-nucleotides of guanine, adenine, hypoxanthine, uracil and cytosine, but never splits nicotinamide mononucleotide, thiamine monophosphate, ribose 5-monophosphate and flavin mononucleotide. 3) The enzyme seems to have specially high affinity for 5′-AMP. 4) The enzyme activity is accelerated by addition of Co⁺⁺ and Ni⁺⁺, but inhibited by Ag⁺, Cu⁺⁺, EDTA, I₂ and N-bromosuccimide. Mg⁺⁺, KCN, NaF and thiol reagents except p-chloromercuribenzoate have no effects. 5) Nucleosides have inhibitory effects, among which adenosine is most effective inhibitor. 6) The activity is reduced up to 30% by dialysis against 1 mm EDTA solution, and the reduced activity is completely reactivated by addition of Co⁺⁺ or Ni⁺⁺, but not by Mn⁺⁺ or Mg⁺⁺.     

Studies on methanol-oxidizing bacteria

Three methanol-oxidizing bacteria were isolated by enrichment culture technique from soil. Two of them werePseudomonas spp. The third one was obligate methylotroph. Their growth characteristics have been described. Cell-suspension experiments withPseudomonas RJ₁ suggest that methanol-, formaldehyde-, and formate-oxidizing enzymes were present. Formate-oxidizing enzyme was detected only fromPseudomonas RJ₁. It had a pH optimum of 7.0 and required nicotinamide adenine dinucleotide (NAD) for activity. Cyanide at 1 × 10⁻⁵ m concentration inhibited the enzyme activity completely.     

Effects induced by rotenone during aerobic growth ofParacoccus denitrificans in continuous culture

Paracoccus denitrificans was grown aerobically in chemostat culture in the presence of rotenone. After 6 to 10 generation times, cells showed an oxygen uptake which was completely rotenone-insensitive after removal of rotenone by washing with bovine serum albumin containing medium.The H⁺/O ratio of these cells for endogenous substrates decreased from about 7.50 to 3.95. The latter ratio was similar to the value obtained for starved cells oxidizing exogenous succinate, indicating that site I phosphorylation was absent in these rotenone-insensitive cells.Membrane particles prepared from these cells showed an 80% decrease in activity of reduced nicotinamide adenine dinucleotide oxidase and reduced nicotinamide adenine dinucleotide-ferricyanide oxidoreductase, while also the kinetic behaviour of the reduced nicotinamide adenine dinucleotide dehydrogenase in the reduced nicotinamide adenine dinucleotide-ferricyanide oxidoreductase assay was changed. Moreover the reduced nicotinamide adenine dinucleotide oxidase activity was practically rotenone-insensitive.Electron paramagnetic resonance spectroscopy on membrane particles from rotenone-insensitive cells at 15 K revealed that the resonance lines atg _z 2.05 andg _y g _x 1.92 arising from iron-sulfur center 2 were undetectable. The intensities of the other electron paramagnetic resonance signals originating from reduced nicotinamide adenine dinucleotide dehydrogenase linked iron-sulfur centers were only slightly diminished.These observations confirm our previous suggestion that site I phosphorylation, rotenone sensitivity and the presence of iron-sulfur center 2 are correlated.Abbreviations EPR electron paramagnetic resonance - BSA bovine serum albumin - CCCP carbonylcyanide m-chlorophenylhydrazone - NAD nicotinamide adenine dinucleotide - NADP nicotinamide adenine dinucleotide phosphate - ATP adenosine triphosphate     

Ribulose bisphosphate carboxylase from Thiobacillus A2. Its purification and properties

Ribulose bisphosphate carboxylase (EC 4.1.1.39) from Thiobacillus A2 has been purified to homogeneity on the basis of polyacrylamide gel electrophoresis and U.V. analysis during sedimentation velocity studies. The enzyme had an optimum pH of about 8.2 with Tris-HCl buffers. The molecular weight was about 521000 with an S _rel. of 16.9. K _m for RuBP was 122 M, for total CO₂ it was 4.17 mM, and for Mg²⁺ 20.0 M. The absolute requirement for a divalent cation was satisfied by Mg²⁺ which was replaceable to a certain extent by Mn²⁺. Activity was not significantly affected by SO ₄ ^2- , SO ₃ ^2- , or S₂O ₃ ^2- at 1.0 mM. At this concentration S^2- caused a 27% stimulation. All mercurials tested were inhibitory. pHMB was the most potent causing about 60% inhibition at 0.01 mM. This inhibition was reversible by low concentrations of cysteine. Cyanide was also inhibitory. Its mode of inhibition with respect to RuBP was un-competitive and with a K _i of 20 M. Lost activity could be restored partially by GSH or Cu²⁺. Although azide at the concentration tested had no significant effect on enzyme activity, 2,4-dinitrophenol at 1.0 mM caused 91% inhibition. Finally, activity was also affected by energy charge.Abbreviations ATP adenosine-5-triphosphate - GAPDH glyceraldehyde phosphate dehydrogenase - GSH (reduced) glutathione - G6P glucose-6-phosphate - NAD⁺ nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - pHMB parahydroxymercuribenzoate - 6PG 6-phosphogluconate - 3-PGA 3-phosphoglycerate - PGK phosphoglyceratekinase - RuBP ribulose-1,5-bisphosphate     

Evidence for two species of glutamate dehydrogenases in Thiobacillus novellus

When grown autotrophically in a thiosulfate-mineral salts medium, cells of the facultative chemoautotrophic bacterium, Thiobacillus novellus, produced two distinct glutamate dehydrogenases, one specific for nicotinamide adenine dinucleotide phosphate (NADP) and the other specific for nicotinamide adenine dinucleotide (NAD). When glutamate was supplied exogenously as the sole carbon source, the NAD-specific glutamate dehydrogenase was fully induced. Lower levels of the enzyme were found in bacteria grown in l-arginine, l-alanine, glucose, glycerol, lactate, citrate, or succinate. Arginine, histidine, and aspartate, on the other hand, caused a marked repression of the NADP-specific glutamate dehydrogenase activity. The NAD-dependent glutamate dehydrogenase was allosteric. Adenosine-5'-monophosphate and adenosine-5'-diphosphate acted as positive effectors. Both glutamate dehydrogenases were purified about 250-fold and were shown to be distinct protein with different physical properties.     

Purification and partial characterization of NAD aminohydrolase from Aspergillus oryzae NRRL447

Aspergillus oryzae aminohydrolase free acid phosphodiesterase catalyzes nicotinamide adenine dinucleotide to deamino-NAD and ammonia. The enzyme was purified to homogeneity by a combination of acetone precipitation, anion exchange chromatography and gel filtration chromatography. The enzyme was purified 230.5 fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band of MW 94 kDa. The enzyme displayed maximum activity at pH 5 and 40 °C with NAD as substrate. The enzyme activity appeared to be stable up to 40 °C. The enzyme activity was enhanced slightly by addition of Na⁺ and K⁺, whereas inhibited strongly by addition of Ag⁺, Mn²⁺, Hg²⁺ and Cu²⁺ to the reaction mixtures. The enzyme hydrolyzes several substrates, suggesting a probable non-specific nature. The enzyme catalyzes the hydrolytic cleavage of amino group of NAD, adenosine, AMP, CMP, GMP, adenosine, cytidine and cytosine to the corresponding nucleotides, nucleosides or bases and ammonia. The substrate concentration–activity relationship is the hyperbolic type and the apparent K_m and K_cat for the tested substrates were calculated.     

Regulation of biosynthesis ofN-glycolylneuraminic acid-containing glycoconjugates: characterization of factors required for NADH-dependent cytidine 5′monophosphate-N-acetylneuraminic acid hydroxylation

The hydroxylation of CMP-NeuAc has been demonstrated to be carried out by several factors including the soluble form of cytochromeb ₅. In the present study, mouse liver cytosol was subjected to ammonium sulfate fractionation and cellulose phosphate column chromatography for the separation of two other essential fractions participating in the hydroxylation. One of the fractions, which bound to a cellulose phosphate column, was able to reduce the soluble cytochromeb ₅, using NADH as an electron donor. The other fraction, which flowed through the column, was assumed to contain the terminal enzyme which accepts electrons from cytochromeb ₅, activates oxygen, and catalyses the hydroxylation of CMP-NeuAc. Assay conditions for the quantitative determination of the terminal enzyme were established, and the activity of the enzyme in several tissues of mouse and rat was measured. The level of the terminal enzyme activity is associated with the expression ofN-glycolylneuraminic acid in these tissues, indicating that the expression of the terminal enzyme possibly regulates the overall velocity of CMP-NeuAc hydroxylation.Abbreviations CMP cytidine 5-monophosphate - NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - NADH reduced nicotinamide adenine dinucleotide - NADPH reduced nicotinamide adenine dinucleotide phosphate - DTT dithiothreitol     

Ornithine dissimilation byTreponema denticola

Treponema denticola convertedl-ornithine, a product ofl-arginine catabolism, to putrescine via a decarboxylation reaction and to proline via a deamination reaction. Ornithine decarboxylation byT. denticola extracts was stimulated by pyridoxal 5′-phosphate. In the absence of pyridoxal 5′-phosphate, (NH₄)₂SO₄-fractionated extracts converted ornithine to proline and ammonia. This activity was not stimulated by α-keto acids, nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide or ADP. Neither ornithine δ-transaminase (l-ornithine: 2-oxoacid aminotransferase, EC 2.6.1.13) nor Δ¹ reductase [l-proline: NAD(P) 5-oxidoreductase, EC 1.5.1.2.] activity was detectable in cell extracts. These results indicate that formation of proline from ornithine inT. denticola is catalyzed by an enzyme system analogous to the ornithine cyclase (deaminating) ofClostridium sporogenes. Exogenous ornithine inhibited the growth ofT. denticola. Thus, in addition to generating putrescine and proline, the ornithine dissimilatory pathways may serve to prevent accumulation of inhibitory concentrations of ornithine in the spirochete's environment.