Methylthioadenosine Nucleosidase Research Articles

5′‐Methylthioadenosine nucleosidase and 5‐methylthioribose kinase activities in relation to stress‐induced ethylene biosynthesis

The activities of 5’‐methylthioadenosine (MTA) nucleosidase (EC 2.2.2.28) and 5‐methylthioribose (MTR) kinase (EC 2.7.1.100) were related to changes in ethylene biosynthesis in tomato (Lycopersicon esculentum Mill. cv. Rutgers) and cucumber (Cucumis sativus Mill. cv. Poinsett 76) fruit following wounding and chemically induced stresses. Stress ethylene formation in wounded tomato and cucumber tissue continued to increase after wounding, reached its peak by 3h, and then declined. The activities of MTA nucleosidase and MTR kinase increased parallel to stress ethylene in both tissues. At peak ethylene formation, MTA and MTR kinase activities were 2‐ to 4‐fold higher in wounded than in intact tissue. Wounded, mature‐green tomato tissue treated with specific inhibitors of MTA nucleosidase and MTR kinase showed a significant reduction in the activities of these enzymes, which was concomitant with a decline in stress ethylene biosynthesis. When mature‐green tomato discs were infiltrated with [14CH3] MTA and wounded, radioactive MTR and methionine were formed. Incubation of mature‐green tomato discs with Cu2+ and Li+ in the presence of kinetin increased ethylene biosynthesis. MTA nucleosidase activity was higher than that of the control in the presence of Cu2+ but not in the presence of Li+, while MTR kinase activity was lower than that of the control in both Cu2+ and Li+ treatments. Data indicate that MTA nucleosidase and MTR kinase are required for wound‐induced ethylene biosynthesis but not for chemical stress‐induced ethylene by Cu2+ or Li+ treatments.

5'-Methylthioadenosine nucleosidase and 5-methylthioribose kinase activities in relation to stress-induced ethylene biosynthesis

5'-Methylthioadenosine nucleosidase and 5-methylthioribose kinase activities in relation to stress-induced ethylene biosynthesis

Selective killing of Klebsiella pneumoniae by 5-trifluoromethylthioribose. Chemotherapeutic exploitation of the enzyme 5-methylthioribose kinase.

5'-Deoxy-5'-methylthioadenosine (MTA), an important intermediate in methionine recycling, can be metabolized by one of two mechanisms that appear to be mutually exclusive. In human cells, MTA is degraded in one step to adenine and 5-methylthioribose 1-phosphate (MTR-1-P) via MTA phosphorylase. In contrast, certain microbes metabolize MTA in two steps: first to 5-methylthioribose (MTR) followed by conversion to MTR-1-P. The enzymes involved in this two-step conversion are MTA nucleosidase and MTR kinase. In both cases, MTR-1-P is subsequently recycled to methionine. Because MTR kinase is "unique" to microbes (it is also found in plant tissue) and since it is essential to microbial methionine salvage, we hypothesized that MTR kinase is a promising target for chemotherapeutic exploitation. We demonstrate that 5-trifluoromethylthioribose (TFMTR), a structural analog of MTR, is a potent inhibitor of the MTR kinase-containing organism Klebsiella pneumoniae. TFMTR not only inhibits the growth of K. pneumoniae in a dose-dependent manner (50% inhibition at approximately 40 nM) but also competitively inhibits MTR kinase activity (Ki approximately 7 microM). Furthermore, TFMTR is shown to be a substrate for MTR kinase (Km = 1.7 microM), suggesting that the drug could be converted to toxic products (e.g. trifluoromethionine or carbonothionic difluoride) in enzyme-containing organisms. Structural analogs of MTR represent a new class of compounds with the potential for treating diseases caused by MTR kinase-containing microorganisms.

Interrelationship of Polyamine and Ethylene Biosynthesis during Avocado Fruit Development and Ripening

Concentrations of polyamines (PA) and the activities of the PA-synthesizing enzymes ornithine decarboxylase (ODC) and arginine decarboxylase (ADC) extracted from the mesocarp tissue of avocado (Persea americana Mill, cv ;Simmonds') fruits at different stages of development were compared with DNA content and the activities of 5'-methylthioadenosine (MTA) nucleosidase and 5-methylthioribose (MTR) kinase. Putrescine, spermidine, and spermine were at their peak concentrations during the early stages of fruit development (362, 201, and 165 nanomoles per gram fresh weight, respectively, at 15 days from full bloom), then declined to 30% or less at full maturity. Agmatine showed only a slight change in concentration throughout the fruit development. The activity of ODC, which was low during flowering (8 nmoles per milligram protein per hour), increased more than threefold during the first 2 months then declined at the later stages of fruit development, while ADC activity showed only a slight increase. DNA content followed a similar pattern of change as that of PA and ODC. The decline in DNA and ODC activity suggest a lack of correlation between cell proliferation and PA at the later stages of the avocado fruit development. It is also possible that any cell division which may take place during the latter stages of the fruit development is not sufficient to alter the pattern of PA biosynthesis. MTA nucleosidase and MTR kinase activities increased during the first 15 days of fruit development followed by a slight decline at 60 and 90 days from full bloom. At 120 days (1 month before full maturity) both MTA nucleosidase and MTR kinase activities increased significantly. During maximum ethylene synthesis, MTA nucleosidase and MTR kinase activities were approximately fivefold and eightfold, respectively, higher than during maximum PA synthesis. The data indicate that the MTA molecules produced during PA and ethylene synthesis are actively metabolized to MTR and MTR-1-P, the two intermediates involved in the regeneration of S-adenosylmethionine from MTA. The data also suggest that the PA and ethylene biosynthetic pathways are not actively competing for the same substrates at any given stage of the avocado fruit development and ripening.

Inhibition of the methionine cycle enzymes

Inhibition of the enzymes involved in the production of 1-aminocyclopropane-1-carboxylic acid (ACC) and the subsequent salvage of methionine from 5′-methylthioadenosine (MTA) was studied. Possible product inhibition of ACC synthase, which converts S-adenosylmethionine (SAM) to ACC and MTA, and MTA nucleosidase, which hydrolyses MTA to 5-methylthioribose (MTR) and adenine, was investigated. ACC synthase was weakly inhibited by MTA ( K i = 0.2mM). MTA nucleosidase was inhibited by adenine competitively ( K i = 40μM), but not by MTR. Some analogues of the enzymes' substrates were inhibitory. ACC synthase was strongly and competitively inhibited by sinefungin, a SAM analogue ( K i = 2μM); MTA nucleosidase was inhibited by various MTA analogues, including 5′-chloroformycin, 5′-chloroadenosine, and 5′-ethylthioadenosine. The conversion of MTR to methionine in avocado extract was inhibited by the MTR analogues 5-chlororibose and 5-ethylthioribose, which exert their inhibitory effects by inhibiting MTR kinase. The capacity to convert MTR to methionine in ripening apple tissue appears to be ample; thus, this conversion does not appear to be a limiting factor of ethylene production.

Escherichia coli S-adenosylhomocysteine/5′-methylthioadenosine nucleosidase. Purification, substrate specificity and mechanism of action

S-Adenosylhomocysteine/5'-methylthioadenosine nucleosidase (EC 3.2.2.9) was purified to homogeneity from Escherichia coli to a final specific activity of 373 mumol of 5'-methylthioadenosine cleaved/min per mg of protein. Affinity chromatography on S-formycinylhomocysteine-Sepharose is the key step of the purification procedure. The enzyme, responsible for the cleavage of the glycosidic bond of both S-adenosylhomocysteine and 5'-methylthioadenosine, was partially characterized. The apparent Km for 5'-methylthioadenosine is 0.4 microM, and that for S-adenosylhomocysteine is 4.3 microM. The maximal rate of cleavage of S-adenosylhomocysteine is approx. 40% of that of 5'-methylthioadenosine. Some 25 analogues of the two naturally occurring thioethers were studied as potential substrates or inhibitors of the enzyme. Except for the analogues modified in the 5'-position of the ribose moiety or the 2-position of the purine ring, none of the compounds tested was effective as a substrate. Moreover, 5'-methylthioformycin, 5'-chloroformycin, S-formycinylhomocysteine, 5'-methylthiotubercidin and S-tubercidinylhomocysteine were powerful inhibitors of the enzyme activity. The results obtained allow the hypothesis of a mechanism of enzymic catalysis requiring as a key step the protonation of N-7 of the purine ring.

5′-Methylthioadenosine Nucleosidase and 5-Methylthioribose Kinase Activities and Ethylene Production during Tomato Fruit Development and Ripening

5'-Methylthioadenosine (MTA) nucleosidase and 5-methylthioribose (MTR) kinase activities were measured in crude extracts of tomato fruits (Lycopersicon esculentum Mill cv Rutgers) during fruit development and ripening. The highest activity of MTA nucleosidase (1.2 nanomoles per milligram protein per minute) was observed in small green fruits. The activity decreased during ripening; at the overripe stage only 6.5% of the peak activity remained. MTR kinase activity was low at the small green stage and increased thereafter until it reached peak activity at the breaker stage (0.7 nanomoles per milligram protein per minute) followed by a sharp decline at the later stages of fruit ripening. 1-Amino-cyclopropane-1-carboxylic acid (ACC) levels peaked at the red stage, while ethylene reached its highest level at the light-red stage. Several analogs of MTA and MTR were tested as both enzyme and ethylene inhibitors. Of the MTA analogs examined for their ability to inhibit MTA nucleosidase, 5'-chloroformycin reduced enzyme activity 89%, whereas 5'-chloroadenosine, 5'-isobutylthioadenosine, 5'-isopropylthioadenosine, and 5'-ethylthioadenosine inhibited the reaction with MTA by about 40%. 5'-Chloroformycin and 5'-chloroadenosine inhibited ethylene production over a period of 24 hours by about 64 and 42%, respectively. Other analogs of MTA were not effective inhibitors of ethylene production, whereas aminoethoxyvinylglycine showed a 34% inhibition over the same period of time. Of the MTR analogs tested, 5-isobutylthioribose was the most effective inhibitor of both MTR-kinase (41%) and ethylene production (35%).

Adenine and adenosine metabolizing enzymes in cell-free extracts from Euglena gracilis

1. 1. Activities of the following enzymes involved in adenine and adenosine metabolism were found in cell-free extracts from Euglena gracilis: acid phosphatase (EC 3.1.3.2), 5′-methylthioadenosine phosphorylase (EC 2.4.2.-), adenine deaminase (EC 3.5.4.2), adenine phosphoribosyltransferase (EC 2.4.2.7) and adenosine kinase (EC 2.7.1.20). 2. 2. The activities occurred both in heterotrophic and photoautotrophic cells and their levels did not change during light-induced chloroplast development. 3. 3. Neither S- adenosylhomocysteinase (EC 3.3.1.1), 5′-methylthioadenosine nucleosidase (EC 3.2.2.9) and nucleoside phosphotransferase (EC 2.7.1.77) nor adenosine degrading enzymes: adenosine deaminase (EC 3.5.4.4), adenosine nucleosidase (EC 3.2.2.7), and purine-nucleoside (adenosine) phosphorylase (EC 2.4.2.1) were found in the Euglena extracts. 4. 4. Comparison of the adenine and adenosine metabolism in Euglena and in other organisms is comprehensively presented. The metabolism in Euglena gracilis differs from that in higher animals and plants.

5'-Methylthioadenosine nucleosidase. Purification and characterization of the enzyme from Lupinus luteus seeds.

5'-Methylthioadenosine nucleosidase (EC 3.2.2.9), the enzyme which catalyzes hydrolytic cleavage of 5'-methylthioadenosine with the formation of adenine and 5'-methylthioribose, has been purified to homogeneity from Lupinus luteus seeds. The nucleosidase has a native molecular weight of 62 000 and consists of two identical subunits, as judged by gel filtration and dodecylsulfate/polyacrylamide gel electrophoresis. The nucleosidase exhibits highest specificity towards the natural substrate with a Km of 4.1 X 10(-7) M for 5'-methylthioadenosine. It does not cleave adenine from S-adenosylhomocysteine. Among the synthetic analogs of 5'-methylthioadenosine tested, eleven compounds appear to be able to substitute as substrates. Furthermore, the enzyme can liberate hypoxanthinine from six inosyl (deaminated) derivatives obtained by enzymatic deamination of 5'-methylthioadenosine and its synthetic analogs. The Km for 5'-methylthioinosine is 55 microM, and the maximal velocity about 50-times lower than for 5'-methylthioadenosine. The reaction catalyzed by the nucleosidase can be inhibited by adenine (Ki = 11 microM), 3-deazaadenine (Ki = 19 microM), and 9-erythro(2-hydroxyl-3-nonyl)adenine (ki = 37 microM).

Methylthioadenosine nucleosidase in normal and dystrophic human muscle

Human skeletal muscle homogenate was found to contain a nucleosidase that catalyzes the hydrolysis of 5'-methylthioadenosine, a known inhibitor of many methyl transfer reactions. When the levels of methylthioadenosine nucleosidase in muscle of patients were compared with those of controls, no significant alterations in its activity were noted in patients with various forms of muscular dystrophies, polymyositis and certain denervating diseases.

Methylthioadenosine nucleoside phosphorylase deficiency in methylthio-dependent cancer cells

The cleavage of the methylthio group from methylthioadenosine is shown to involve two enzymes, a nucleosidase which catalyses the phosphorolytic cleavage of methylthioadenosine to yield adenine and 5-methylthioribose-1-phosphate and an enzyme which uses the latter compound as substrate and catalyses the release of the methylthio group as an ether-extractable product. Three malignant murine hematopoietic cell lines which require methylthio group supplementation for proliferation in vitro are shown to lack methylthioadenosine nucleosidase activity while retaining activity of the second enzyme. Four cell lines which are methylthio-independent in vitro contain activity of both enzymes. The data suggest that the requirement for exogenous methylthio groups in certain cells is caused by the block in their biosynthetic pathway imposed by methylthioadenosine nucleoside phosphorylase deficiency. Secondarily, the data suggest that all cells require methylthio or related groups for division.

Studies on phosphate-activated 5′-methylthioadenosine nucleosidase from human placenta

1. 1.Evidence for the presence of 5′-methylthioadenosine nucleosidase in human placenta is reported; the reaction involves the cleavage of the glycosidic bond of the thioether and the release of equimolar amounts of adenine and 5-methylthioribose. 2. 2. The enzyme is moderately stable and has been purified 400-fold in 17% yield: the use of affinity chromatography by Sepharose-Hg has been found particularly advantageous. 3. 3. The enzyme requires reducing agents: only 67% of maximal activity is observable in absence of dithiothreitol and 2-mercaptoethanol. 4. 4. The reaction rate is markedly increased by temperature elevation, exhibiting a maximum at 67°C. From the Arrhenius plot an apparent activation energy of 6390 cal/mole has been calculated. 5. 5. A mol. wt of 95,000 has been estimated: the inhibitory effect of thiol reagents suggests the presence of “essential thiol groups”. 6. 6. The enzyme requires phosphate ions for its activity: the apparent K a for phosphate is 1.5mM. Substrate analogues, such as 5′deoxy-5′-S- adenosyl- l-homocysteine , 5′deoxy-5′-S- adenosyl- l-cysteine , and 5′deoxy-5′-thioethanol-adenosine, do not appreciably influence the reaction rate.

Kinetic properties and the effect of substrate analogues on 5′-methylthioadenosine nucleosidase from Escherichia coli

5′-Methylthioadenosine nucleosidase (EC 3.2.2.-) from Escherichia coli has been purified 220-fold. A molecular weight of 31 000 for the enzyme was estimated from gel filtration on Sephadex G-150. The K m for 5′-methylthioadenosine was 3.1 · 10 −7 M. In addition to 5′-methylthioadenosine, the nucleoside analogues 5′-ethylthioadenosine, 5′-n- propylthioadenosine , and S- adenosylhomocysteine also served as substrates for the enzyme. These substrate analogues acted as competitive inhibitors of the reaction with 5′-methylthioadenosine. The K i values for 5′-ethylthioadenosine, 5′-n- propylthioadenosine , and S- adenosylhomocysteine were determined to be 1.3 · 10 −7 M, 4.6 · 10 −8 M, and 1.92 · 10 −7 M respectively.

Studies on metabolic role of 5'-Methylthioadenosine in Ochromonas malhamensis and other microorganisms.

Several compound containing a thiomethyl group were found to replace vitamin B12 in a protozoan, Ochromonas malhamensis. The order of the effectiveness was as follows: 5'-methylthioadenosine is greater than S-adenosylmethionine is greater than 5-methylthioribose is greater then L-methionine. A similar order was obtained with respect to the permeability of these compounds into the protozoan cells, except for S-adenosylmethionine. 5'-Methylthioadenosine and 5-methylthioribose as well as L-methionine markedly increased the intracellular content of L-methionine. The level of S-adenosylmethionine was also increased by them, but to lesser degree. The thiomethyl group of the compounds was established to be incorporated into S-adenosylmethionine. The metabolic fate of the thiomethyl group of 5'-methylthioadenosine cannot be distinguished from that of L-methionine. A high activity of 5'-methylthioadenosine nucleosidase was detected in the cell-free extracts of the protozoan. These results strongly suggest that 5'-methylthioadenosine would be metabolized to L-methionine would be ocnverted to S-adenosylmethionine. Like L-methionine and vitamin B12, 5'-methylthioadenosine and 5-methylthioribose may play an important role in maintenance of the C-1 pool in Ochromonas malhamensis. Neither 5'-methylthioadenosine nor 5-methylthioribose replaced vitamin B12 in some vitamin B12-requiring bacteria. This result is consistent with the fact that neither compounds was significantly taken up by these bacteria.