Evidence for in vivo synthesis of thiamin triphosphate by cytosolic adenylate kinase in chicken skeletal muscle

We showed previously that cytosolic adenylate kinase (AK1) purified from pig skeletal muscle catalyzes in vitro formation of thiamin triphosphate (TTP) from thiamin diphosphate (TDP) and ADP in addition to ATP formation from ADP [Shikata, H. et al. (1989) Biochem. Int. 18, 933-942]. To obtain evidence for in vivo synthesis of TTP by AK1, changes in TTP content and AK1 activity were determined in chicken skeletal muscle during development after hatching. Thiamin phosphate metabolism in chicken skeletal muscle was also studied. i) An extremely high TTP content, 81% of total thiamin (thiamin plus thiamin phosphates), was detected in the white (fast-twitch) muscle of adult normal chicken (5th to 9th month) compared with a relatively high TTP content of 31% in the red (slow-tonic) muscle. Since approximately equivalent amounts of total thiamin were present in the two types of muscle, the ratio of TTP to TDP was high (5.0) in the white muscle and low (0.41) in the red muscle. ii) Rabbit anti-chicken AK1 antiserum against the purified chicken cytosolic AK1 preparation was obtained. Both AK1 activity and TTP-synthesizing activity in crude cytosol fraction of adult chicken white muscle were inhibited in parallel by the antiserum. iii) In the white muscle of normal chicken, the TTP content and AK1 activity responsible for forming either ATP or TTP were increased in a parallel manner up to day 16 after hatching, after which both remained constant. In the red muscle, on the other hand, both the TTP content and the AK1 activity were low in comparison with those in the white muscle, and were almost constant after hatching.(ABSTRACT TRUNCATED AT 250 WORDS)     

Muscle adenylate kinase catalyzes adenosine 5'-tetraphosphate synthesis from ATP and ADP

Muscle adenylate kinases from rabbit and porcine sources were found by NMR to catalyze the formation of adenosine tetraphosphate (5'AdoP4) from adenosine triphosphate (ATP) and adenosine diphosphate (ADP). The reaction was completely reversible, with an equilibrium constant of approx. 0.1 at 30 degrees C. The synthesis of 5'AdoP4 from ATP and ADP occurred very slowly, taking over 12 h to reach equilibrium under the conditions used in this study. The sources of micromolar concentrations of 5'AdoP4 found in biological tissues is unknown: potentially, the slow adenylate-kinase-catalyzed breakdown and synthesis of 5'AdoP4 may serve as an important regulator of the steady-state concentration of 5'AdoP4 in muscle tissue.     

Enzyme system involved in the synthesis of thiamin triphosphate. I. Purification and characterization of protein-bound thiamin diphosphate: ATP phosphoryltransferase

An enzyme system catalyzing the synthesis of thiamin triphosphate consists of an enzyme (protein-bound thiamin diphosphate:ATP phosphoryltransferase), thiamin diphosphate bound to a macromolecule as substrate, ATP, Mg2+, and a low molecular weight cofactor. This system was established by combining a purified enzyme and an essentially pure, macromolecule-bound substrate prepared from rat livers. This macromolecule was found to be a protein, and the transphosphorylation of thiamin diphosphate to thiamin triphosphate with ATP and enzyme was shown to occur on this macromolecule which binds thiamin diphosphate. Free thiamin, thiamin monophosphate, thiamin diphosphate, and thiamin triphosphate have no effect on this reaction. Thus, the overall reaction is: thiamin diphosphate-protein + ATP in equilibrium thiamin triphosphate-protein + ADP. So-called thiamin diphosphate:ATP phosphoryltransferase (EC 2.7.4.15) activity was not detected in rat brain or liver. The enzyme was extracted from acetone powder of a crude mitochondrial fraction of bovine brain cortex and purified to homogeneity with a 0.6% yield after DEAE-cellulose chromatography, a first gel filtration, hydroxylapatite chromatography, chromatofocusing, and a second gel filtration. The purified enzyme showed a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Its molecular weight was estimated to be 103,000. The pH optimum was 7.5, and the Km was determined to be 6 X 10(-4) M for ATP. ATP was found to be the most effective phosphate donor among the nucleoside triphosphates. Amino acid analysis of the purified enzyme revealed an abundance of glutaminyl, glutamyl, and aspartyl residues. Sulfhydryl reagents inhibited the enzyme reaction. Metals such as Fe2+, Zn2+, Pb2+, and Cu2+ strongly inhibited the activity. The enzyme was unstable, and glycerol (20%) and dithiothreitol (1.0 mM) were found to preserve the enzyme activity.     

Nucleoside triphosphate synthesis catalysed by adenylate kinase is ADP dependent

Adenylate kinase (Adk) that catalyses the synthesis of ADP from ATP and AMP has also been shown to perform an ATP dependent phosphorylation of ribo- and deoxynucleoside diphosphates to their corresponding nucleoside triphosphate; ATP+(d)NDP<-->ADP+(d)NTP. This reaction, suggested to occur by the transfer of the gamma-phosphoryl from ATP to the nucleoside diphosphate, is overall similar to that normally carried out by nucleoside diphosphate kinase (Ndk). Accordingly, Adk was proposed to be responsible for residual Ndk-like activity measured in a mutant strain of Escherichia coli, where the ndk gene was disrupted. We present data supporting a mechanism for the synthesis of nucleoside triphosphates by Adk that unlike the previously suggested mechanism mentioned above are in complete agreement with the current knowledge about the Adk enzyme and its various catalytic properties. We propose that nucleoside triphosphate synthesis occurs by beta-phosphoryl transfer from ADP to any bound nucleoside diphosphate. Our results point to the fact that the proposed Ndk-like mechanism of Adk originated from an erroneous interpretation of data, in that contamination of ATP preparations with AMP and ADP was not taken into account. Our results also address the proposed role of Adk in restoring a normal growth rate of mutant strains of E. coli lacking Ndk. These mutant strains apparently, in spite of a mutator phenotype, are able to synthesise nucleoside triphosphates by alternative pathways to maintain the same growth rate as the wildtype.     

Existence in animal tissues of adenosine triphosphate thiamin diphosphate phosphotransferase [EC 2.7.4.15]

An enzyme which catalyzes the synthesis of thiamin triphosphate from thiamin diphosphate (TDP), thiamindiphosphate kinase (ATP:thiamin diphosphate phosphotransferase) [EC 2.7.4.15], was detected in animal tissues. The enzyme was partially purified (150-fold) from the cytosol fraction of guinea pig brain. The enzyme reaction required free (not protein-bound) TDP, ATP, Mg2+, and a cofactor, which is a low molecular weight and heat-stable compound. The enzyme activity was optimal at pH 11 and at 25 degrees C. A stoichiometric transfer of 32P from [gamma-32P]ATP to TDP was demonstrated. Km values for TDP and ATP were calculated to be 1.1 mM and 10 microM, respectively, and Vmax was 868 nmol/mg of protein/hr. The enzyme was found solely in the cytosol fraction of guinea pig brain and was also detectable in the skeletal muscle and heart. These results provide strong evidence for the existence of TDP kinase in animal tissues.     

Response of nucleoside diphosphate kinase to the adenylate energy charge

The reaction catalyzed by nucleoside diphosphate kinase responds to the energy charge of the adenylate pool. The velocity is maximal at a charge of 1.0, and decreases sharply with a decrease in the charge. This response may control the flow of phosphate from ATP into the other nucleotide pools and thus participate in the regulation of macromolecular synthesis by the energy level of the cell, as reflected in the charge of the adenylate pool.     

Identification of mitochondrial thiamin diphosphate carriers from Arabidopsis and maize

It is currently held that thiamin is made in chloroplasts and converted in the cytosol to the active cofactor thiamin diphosphate (ThDP), and that mitochondria and plastids import ThDP. The organellar transporters that mediate ThDP import in plants have not been identified. Comparative genomic analysis indicated that two members of the mitochondrial carrier family (MCF) in Arabidopsis (At5g48970 and At3g21390) and two in maize (GRMZM2G118515 and GRMZM2G124911) are related to the ThDP carriers of animals and Saccharomyces cerevisiae. Expression of each of these plant proteins in a S. cerevisiae ThDP carrier (TPC1) null mutant complemented the growth defect on fermentable carbon sources and restored the level of mitochondrial ThDP and the activity of the mitochondrial ThDP-dependent enzyme acetolactate synthase. The plant proteins were targeted to mitochondria as judged by dual import assays with purified pea mitochondria and chloroplasts, and by microscopic analysis of the subcellular localization of green fluorescent protein fusions in transiently transformed tobacco suspension cells. Both maize genes were shown to be expressed throughout the plant, which is consistent with the known ubiquity of mitochondrial ThDP-dependent enzymes. Collectively, these data establish that plants have mitochondrially located MCF carriers for ThDP, and indicate that these carriers are highly evolutionarily conserved. Our data provide a firm basis to propagate the functional annotation of mitochondrial ThDP carriers to other angiosperm genomes.     

Properties of the thiamin triphosphate-synthesizing activity catalyzed by adenylate kinase (isoenzyme 1)

Adenylate kinase isozyme 1 (AK1) catalyzes thiamin triphosphate (TTP) formation from thiamin diphosphate (TDP) and ADP. The properties of the TTP-synthesizing activity of purified AK1 from porcine skeletal muscle were studied. The activity was found to require TDP, ADP, and Mg2+, and ATP was only 14.4% as active as ADP. Thiamin monophosphate (TMP) and thiamin were not utilized as substrates. ADP was specific as a phosphate donor; and CDP, UDP, and GDP supported TTP formation at rates less than 1% of that with ADP. Optimal pH and temperature for the TTP-synthesizing activity were 10.0 and 37 degrees C, respectively. The activity showed saturation kinetics for both substrates, and the Km values for TDP and ADP were calculated to be 0.83 mM and 43 microM, respectively. The enzyme catalyzed the reverse reaction (TTP + AMP----TDP + ADP) and stoichiometry between TTP and TDP was demonstrated in the forward and reverse reactions.     

Vanadate catalyzes photocleavage of adenylate kinase at proline-17 in the phosphate-binding loop.

Irradiation of adenylate kinase (AK) from chicken muscle with 300-400-nm light in the presence of 0.25 mM vanadate ion first inactivated the enzyme and then cleaved the polypeptide chain near the NH2 terminus. The addition of the multisubstrate analogue, P1,P5-bis(5'-adenosyl) pentaphosphate, prevented both effects. ATP, but not AMP, blocked both inactivation and cleavage in a saturable manner, suggesting that both effects were due to modification at the ATP-binding site. The polypeptide products of the photocleavage were isolated by HPLC and characterized by amino acid composition, peptide sequencing, and mass spectral analyses. The predominant (greater than 90%) small peptide fragment contained the first 16 amino acids from the amino terminus of the enzyme. The amino terminus of this peptide contained an acetylated serine, and the "carboxy" terminus was modified by a cyclized gamma-aminobutyric acid which originated from photooxidation and decarboxylation of proline-17 by vanadate. Edman sequencing indicated that the majority of the large peptide fragment (Mr approximately 19,500) was amino-terminal blocked, but a small portion was sequenceable starting at either glycine-18 (7%) or serine-19 (2%). These studies indicate that in the ATP-AK complex proline-17 is close to the phosphate chain of ATP but not AMP, consistent with the latest evaluation of nucleotide-binding sites on mitochondrial matrix AK by X-ray crystallography [Diederichs, K., & Schulz, G.E. (1991) J. Mol. Biol. 217, 541-549]. Furthermore, this is the first report that an amino acid other than serine can be involved in vanadate-promoted photocleavage reactions.     

The nucleoside diphosphate kinase of Mycobacterium smegmatis: identification of proteins that modulate specificity of nucleoside triphosphate synthesis by the enzyme

We report the purification and characterization of the enzyme nucleoside diphosphate kinase (Ndk) from Mycobacterium smegmatis . The N-terminus of the enzyme was blocked but an internal sequence showed approx. 70% homology with the same enzymes from Pseudomonas aeruginosa and Escherichia coli . Immobilization of the mycobacterial nucleoside diphosphate kinase on a Sepharose 4B matrix and passing the total cell extract through it revealed four proteins (P₇₀, P₆₅, P₆₀, and P₅₀, respectively) of M _r 70 kDa, 65 kDa, 60 kDa and 50 kDa that were retained by the column. While the proteins of M _r 70 kDa and 50 kDa modulated the activity of Ndk directing it towards GTP synthesis, the 60 kDa protein channelled the specificity of Ndk entirely towards CTP synthesis. The 65 kDa protein modulated the specificity of Ndk directing it entirely towards UTP synthesis. The specificity for such mycobacterial proteins towards NTP synthesis is retained when they are complexed with P. aeruginosa Ndk. We further demonstrate that the P₇₀ protein is pyruvate kinase and that each of the four proteins forms a complex with Ndk and alters its substrate specificity. Given the ubiquitous nature of Ndk in the living cell and its role in maintaining correct ratios of intracellular nucleoside triphosphates, the implications of the occurrence of these complexes have been discussed in relation to the precursor pool for cell wall biosynthesis as well as RNA/DNA synthesis.     

Properties of thiamin triphosphate-synthesizing activity of chicken cytosolic adenylate kinase and the effect of adenine nucleotides

Cytosolic adenylate kinase synthesis thiamin triphosphate (TTP) from thiamin diphosphate (TDP) in vitro by a reversible reaction: TDP + ADP Mg2+ in equilibrium TTP + AMP. The backward (TTP----TDP) reaction rate was 3-times faster than the forward (TDP----TTP) reaction rate when all the substrate concentrations were 0.1 mM. This property of TTP-synthesizing activity of the enzyme did not explain the fact that the [TTP]/[TDP] molar ratio determined in chicken white skeletal muscle is 5.0 (Miyoshi, K., Egi, Y., Shioda, T. and Kawasaki, T. (1990) J. Biochem. 108, 267-270). To solve this problem, we have studied the properties of TTP-synthesizing activity of the purified recombinant chicken cytosolic adenylate kinase preparation and the effect of adenine nucleotides, especially of ATP. The backward reaction of the TTP synthesis did not proceed in the presence of 8.8 mM ATP, a physiological concentration in chicken white skeletal muscle, while the forward reaction proceeded at a reduced rate. The [TTP]/[TDP] ratio found after a long incubation period was 3.0 and 0.7, respectively, in the presence and absence of 8.8 mM ATP. These results indicate that the high [TTP]/[TDP] molar ratio found in chicken white muscle was demonstrated in vitro by the purified chicken cytosolic adenylate kinase and support in vivo TTP synthesis by this enzyme.     

The genes and enzymes involved in the biosynthesis of thiamin and thiamin diphosphate in yeasts

Thiamin (vitamin B1) is an essential molecule for all living organisms. Its major biologically active derivative is thiamin diphosphate, which serves as a cofactor for several enzymes involved in carbohydrate and amino acid metabolism. Important new functions for thiamin and its phosphate esters have recently been suggested, e.g. in gene expression regulation by influencing mRNA structure, in DNA repair after UV illumination, and in the protection of some organelles against reactive oxygen species. Unlike higher animals, which rely on nutritional thiamin intake, yeasts can synthesize thiamin de novo. The biosynthesis pathways include the separate synthesis of two precursors, 4-amino-5-hydroxymethyl-2-methylpyrimidine diphosphate and 5-(2-hydroxyethyl)-4-methylthiazole phosphate, which are then condensed into thiamin monophosphate. Additionally, yeasts evolved salvage mechanisms to utilize thiamin and its dephosphorylated late precursors, 4-amino-5-hydroxymethyl-2-methylpyrimidine and 5-(2-hydroxyethyl)-4-methylthiazole, from the environment. The current state of knowledge on the discrete steps of thiamin biosynthesis in yeasts is far from satisfactory; many intermediates are postulated only by analogy to the much better understood biosynthesis process in bacteria. On the other hand, the genetic mechanisms regulating thiamin biosynthesis in yeasts are currently under extensive exploration. Only recently, the structures of some of the yeast enzymes involved in thiamin biosynthesis, such as thiamin diphosphokinase and thiazole synthase, were determined at the atomic resolution, and mechanistic proposals for the catalysis of particular biosynthetic steps started to emerge. Paper authored by participants of the international conference: XXXIV Winter School of the Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian University, Zakopane, March 7–11, 2007, “The Cell and Its Environment”. Publication cost was partially covered by the organisers of this meeting.     

Properties and functions of the thiamin diphosphate dependent enzyme transketolase

This review highlights recent research on the properties and functions of the enzyme transketolase, which requires thiamin diphosphate and a divalent metal ion for its activity. The transketolase-catalysed reaction is part of the pentose phosphate pathway, where transketolase appears to control the non-oxidative branch of this pathway, although the overall flux of labelled substrates remains controversial. Yeast transketolase is one of several thiamin diphosphate dependent enzymes whose three-dimensional structures have been determined. Together with mutational analysis these structural data have led to detailed understanding of thiamin diphosphate catalysed reactions. In the homodimer transketolase the two catalytic sites, where dihydroxyethyl groups are transferred from ketose donors to aldose acceptors, are formed at the interface between the two subunits, where the thiazole and pyrimidine rings of thiamin diphosphate are bound. Transketolase is ubiquitous and more than 30 full-length sequences are known. The encoded protein sequences contain two motifs of high homology; one common to all thiamin diphosphate-dependent enzymes and the other a unique transketolase motif. All characterised transketolases have similar kinetic and physical properties, but the mammalian enzymes are more selective in substrate utilisation than the nonmammalian representatives. Since products of the transketolase-catalysed reaction serve as precursors for a number of synthetic compounds this enzyme has been exploited for industrial applications. Putative mutant forms of transketolase, once believed to predispose to disease, have not stood up to scrutiny. However, a modification of transketolase is a marker for Alzheimer’s disease, and transketolase activity in erythrocytes is a measure of thiamin nutrition. The cornea contains a particularly high transketolase concentration, consistent with the proposal that pentose phosphate pathway activity has a role in the removal of light-generated radicals.     

The phosphorylation of 5′-oligoadenylic acids by adenylate kinase and adenosine triphosphate

1. This paper reports studies on the metabolism of bone from normal chicks and from chicks with vitamin D-deficiency rickets. Both in vitro and in vivo there was an increased incorporation of [(14)C]proline into collagen hydroxyproline by rachitic bone. The proportion of the collagen that was soluble in cold salt solutions was greater with the rachitic bone. These results show that in rickets there is an increased synthesis of bone collagen, but they do not provide any evidence of a defect in the maturation of collagen. 2. Rachitic bone incubated aerobically in vitro consumed more glucose and released more lactate than normal bone. Bone from rachitic chicks treated with vitamin D 48hr. previously had rates of glycolysis that were nearly normal. Though we were unable to show any direct action of vitamin D in vitro, we consider that vitamin D probably has a direct action on bone, possibly related to matrix biosynthesis.     

Nucleoside diphosphate kinase from lily pollen

Nucleoside diphosphate kinase was a predominantly soluble enzymein lily pollen (Lilium longiflorum, var. Ace), and very littleenzyme activity was associated with the mitochondrial fraction.GTP was the preferred substrate when ADP was the second substrate.The enzyme was purified 18-fold to a specific activity of 7µmoles/min/mg protein. ¹This study was supported by grant GB-8764 from the U.S. NationalScience Foundation. (Received October 5, 1970; )     

Aspergillus niger catalyzes the synthesis of flavonoids from chalcones

Abstract

Flavonoids, which have many biological activities and have been widely used in nature, can be artificially synthesized. However, regioselective cyclization of chalcones is difficult by chemical methods. In this study, we demonstrated that Aspergillus niger is capable of cyclizing chalcones to flavanones, affording a mimic of plant biosynthetic processes. Chalcones 1–6 were biotransformated to the modified chalcones 8–14 and to the flavanones 15–27. The biotransformation showed that enzymatic cyclization and demethylation occurred during the first days of biotransformation; in contrast, hydroxylation is a later process. With a longer culturing time, it is possible to obtain more hydroxylated flavanones with excellent yields.     

NMR analysis of covalent intermediates in thiamin diphosphate enzymes

Enzymic catalysis proceeds via intermediates formed in the course of substrate conversion. Here, we directly detect key intermediates in thiamin diphosphate (ThDP)-dependent enzymes during catalysis using (1)H NMR spectroscopy. The quantitative analysis of the relative intermediate concentrations allows the determination of the microscopic rate constants of individual catalytic steps. As demonstrated for pyruvate decarboxylase (PDC), this method, in combination with site-directed mutagenesis, enables the assignment of individual side chains to single steps in catalysis. In PDC, two independent proton relay systems and the stereochemical control of the enzymic environment account for proficient catalysis proceeding via intermediates at carbon 2 of the enzyme-bound cofactor. The application of this method to other ThDP-dependent enzymes provides insight into their specific chemical pathways.