Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT02288 (NADH)
 21,914 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An enzyme system of Mycobacterium smegmatis catalyzing the elongation of medium-chain fatty acids with acetyl-CoA was obtained free from de novo fatty acid synthetase by ammonium sulfate fractionation. The system was resolved by gel filtration and DEAE-cellulose chromatography into three fractions, all of which were required for reconstitution of the elongation activity. The three fractions were highly purified enoyl-CoA hydratase, highly purified 3-hydroxyacyl-CoA dehydrogenase, and a fraction containing both enoyl-CoA reductase and thiolase. The reconstituted system was avidin-insenstive, required NADH as a sole hydrogen donor, and was sensitive to pCMB, but not to N-ethylmaleimide or monoiodoacetate. Decanoyl-CoA and octanoyl-CoA were the best primers for the elongation system. When decanoyl-CoA was used as the primer, the major product was found to be a lauroyl derivative (probably lauroyl-CoA). Evidence was obtained suggesting that acyl-CoA dehydrogenase, catalyzing the first step of beta-oxidation, was not functional in the elongation system.
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PMID:Acetyl-CoA-dependent elongation of fatty acids in Mycobacterium smegmatis. 2 Nov 75

beta-Hydroxybutyrate dehydrogenase (EC 1.1.1.30) was purified 145-fold from Mycobacterium phlei ATCC354 by ammonium sulphate fractionation and DEAE-cellulose chromatography. The pH optima for oxidation and reduction reactions were 8.4 and 6.8 respectively. The purified enzyme was specific for NAD, NADH, acetoacetate and D(-)-beta-hydroxybutyrate. Km values for DL-beta-hydroxybutyrate and NAD were 7.4 mM and 0.66 mM respectively. The enzyme was inactivated by mercurial thiol inhibitors and by heat, but could be protected by NADH, Ca2+ and partially by Mn2+. The enzyme did not require metal ions and was insensitive to EDTA, glutathione, dithiothreitol, beta-mercaptoethanol and cysteine.
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PMID:Purification and properties of beta-hydroxybutyrate dehydrogenase from Mycobacterium phlei ATCC354. 2 83

The kinetics of reduction of the b-type cytochromes in the electron transport particles (ETP) from Mycobacterium phlei were studied with nicotinamide adenine dinucleotide, reduced form (NADH) or succinate as electron donors. There appeared to be three active cytochromes b in the ETP,bS563 and bS559, which were reducible by either substrate, and bN563, which was reducible by NADH but not by succinate. In the presence of adenosine 5'-triphosphate, a substantial increase in b563 reduction was observed with succinate at anaerobiosis. This was followed by a decrease in absorption. Adenosine 5'-triphosphate did not effect an increase in cytochrome b563 reduction at transition with NADH, but the occurrence of a secondary decrease in absorption was reflected in a decrease in total enzymatic reduction. The adenosine 5'-triphosphate effect was altered in trypsin-treated ETP, and abolished by uncoupling agents or by removal of the coupling factor-latent adenosine triphosphatase. In the presence of a supernatant fraction obtained during the preparation of the ETP, b563 reduction with succinate was greatly increased. A smaller increase was observed with NADH. Cytochrome b reduction was also studied in ETP inhibited by 2-n-nonylhydroxyquinoline-N-oxide, which appears to inhibit at bS563. On the basis of these data the interrelationships among the b-type cytochromes can be described in relation to the M. phlei electron transport chain.
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PMID:Multiple forms of cytochrome b in Mycobacterium phlei: kinetics of reduction. 16 77

Particles from Mycobacterium lepraemurium catalysed the oxidation of NADH with oxygen as the terminal electron acceptor. The preparations contained cytochromes of the a + a3'b and c types, as well as CO-binding pigments. The NADH oxidase activity was sensitive to inhibitors of the flavoprotein system as well as to HQNO and antimycin A. In addition, a cytochrome oxidase sensitive to cyanide was also present. The system was inhibited by the thiol-binding agent, PCMB, and thus indicated the involvement of sulphydryl group in the enzymatic oxidation of NADH. The sensitivity of the NADH oxidase system to all the inhibitors of the respiratory chain and the effect of these inhibitors on the absorption spectra suggested that cytochromes of the b, c, a + a3 types are involved in the transfer of electrons in NADH oxidation.
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PMID:Oxidation of reduced nicotinamide adenine dinucleotide by particles from Mycobacterium lepraemurium. 17 40

The mechanism of action of isoniazid (INH) on saprophytic and atypical mycobacteria is thought to be different from that on Mycobacterium tuberculosis because higher concentrations are required to be effective in these species. In this investigation, M. phlei was inhibited by INH at a concentration of 25 mug/ml. Benzoic acid hydrazide (BZH) and nicotinic acid hydrazide (NAH) were inhibitory at levels of 300 and 500 mug/ml, respectively. Inhibition by these compounds was not inoculum dependent. An isolated M. phlei mutant resistant to 100 mug of INH per ml (Inh(r)) was inhibited by INH only at concentrations about equal to those inhibitory for BZH and NAH. When NAH and BZH were below their minimal inhibitory concentrations, INH inhibition was antagonized. Hence, there appears to be a single target site for INH in mycobacteria with different affinities for various hydrazide analogs of INH. The increased inhibitory levels required for the atypical and saprophytic species are due to a decreased affinity of the target site for INH in these species. INH also inhibited both the oxidized nicotinamide adenine dinucleotide (NAD(+)) and adenosine 5'-monophosphate stimulation of reduced NAD (NADH) oxidase activity associated with the M. phlei and M. tuberculosis H(37)R(a) electron transport particles. INH did not reverse the NAD(+) stimulation of oxidase activity in the Inh(r) strain of M. phlei. No direct inhibitory effect of INH on NADH oxidase activity was observed. Incubation of M. phlei electron transport particles at 0 degrees C with INH resulted in a dramatic loss of oxidase activity which could have been prevented if NAD(+) were present. However, INH had no effect upon the NADH oxidase when stored with electron transport particles isolated from the Inh(r) strain. Therefore, INH inhibition of regulation and/or stabilization of the electron transport pathway by NAD(+) or adenosine 5'-monophosphate may account, in part, for the lethal action of the drug on mycobacteria.
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PMID:Specificity of isoniazid on growth inhibition and competition for an oxidized nicotinamide adenine dinucleotide regulatory site on the electron transport pathway in Mycobacterium phlei. 19 85

NAD+ had a biphasic effect on the NADH oxidase activity in electron transport particles from Mycobacterium phlei. The oxidase was inhibited competitively by NAD+ at concentrations above 0.05 mM. NAD+ in concentrations from 0.02 to 0.05 mM resulted in maximum stimulation of both NADH oxidation and oxygen uptake with concentrations of substrate both above and below the apparent K-M. Oxygen uptake and cyanide sensitivity indicated that the NAD+ stimulatory effect was linked to the terminal respiratory chain. The stimulatory effect was specific for NAD+. NAD+ was also specific in protecting the oxidase during heating at 50 degrees and against inactivation during storage at 0 degrees. NAD+ glycohydrolase did not affect stimulation nor heat protection of the NADH oxidase activity if the particles were previously preincubated with NAD+. Binding studies revealed that the particles bound approximately 3.6 pmol of [14C1NAD+ per mg of electron transport particle protein. Although bound NAD+ represented only a small fraction of the total added NAD+ necessary for maximal stimulation, removal of the apparently unbound NAD+ by Sephadex chromatography revealed that particles retained the stimulated state for at least 48 hours. Further addition of NAD+ to stimulated washed particles resulted in competitive inhibition of oxidase activity. Desensitization of the oxidase to the stimulatory effect of NAD+ was achieved by heating the particles at 50 degrees for 2 min without appreciable loss of enzymatic activity. Kinetic studies indicated that addition of NADH to electron transport particles prior to preincubation with NAD+ inhibited stimulation. In addition, NADH inhibited binding of [14C]NAD+. The utilization of artificial electron acceptors, which act as a shunt of the respiratory chain at or near the flavoprotein component, indicated that NAD+ acts as at the level of the NADH dehydrogenase at a site other than the catalytic one resulting in a conformational change which causes restoration as well as protection of oxidase activity.
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PMID:Effect of nicotinamide adenine dinucleotide on the membrane-associated reduced nicotinamide adenine dinucleotide oxidase of Mycobacterium phlei. 23 63

3-Hydroxyacyl-CoA dehydrogenase [EC 1.1.1.35] was purified 100-fold to homogeneity from crude extracts of Mycobacterium smegmatis, using ammonium sulfate fractionation, gel filtration, and chromatography on DEAE-cellulose, hydroxyapatite, and NAD-Sepharose 4B columns. Its molecular weight was estimated to be 50,300 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NADH acted twelve times more efficiently than NADPH as an electron donor for the reduction of 3-ketoacyl-CoA, and there was strict substrate stereospecificity (L form) in the oxidation of 3-hydroxyacyl-CoA. The pH optimum depended upon the direction of reaction, i.e., 6.0 for the oxidation of NADH and 9--10 for the reduction of NAD. The Km values for different thioesters of acetoacetate, i.e., esters of CoA, pantetheine, and acetyl-cysteamine were determined to be 0.036, 1.19, and 44.4 mM, respectively. Antibodies raised against the dehydrogenase of M. smegmatis strongly inhibited the enzyme activity, but did not affect the corresponding dehydrogenase of pig heart. The antibodies were found to inhibit the acetyl-CoA dependent elongation of fatty acids by the crude extract of M. smegmatis. These findings, together with those on the reconstitution of the elongation activity reported previously (Shimakata, T., Fujita, Y., & Kusaka, T. (1977) J. Biochem. 82, 725-732) indicate that 3-hydroxyacyl-CoA dehydrogenase is involved in the acetyl-CoA dependent elongation of fatty acids in M. smegmatis.
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PMID:Purification and characterization of 3-hydroxyacyl-CoA dehydrogenase of Mycobacterium smegmatis. 52 33

Alkene monooxygenase, a multicomponent enzyme system which catalyzes the epoxidation of short-chain alkenes, is induced in Mycobacterium strain E3 when it is grown on ethene. We purified the NADH reductase component of this enzyme system to homogeneity. Recovery of the enzyme was 19%, with a purification factor of 920-fold. The enzyme is a monomer with a molecular mass of 56 kDa as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is yellow-red with absorption maxima at 384, 410, and 460 nm. Flavin adenine dinucleotide (FAD) was identified as a prosthetic group at a FAD-protein ratio of 1:1. Tween 80 prevented irreversible dissociation of FAD from the enzyme during chromatographic purification steps. Colorimetric analysis revealed 2 mol each of iron and acid-labile sulfide, indicating the presence of a [2Fe-2S] cluster. The presence of this cluster was confirmed by electron paramagnetic resonance spectroscopy (g values at 2.011, 1.921, and 1.876). Anaerobic reduction of the reductase by NADH resulted in formation of a flavin semiquinone.
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PMID:Purification and properties of the NADH reductase component of alkene monooxygenase from Mycobacterium strain E3. 131 34

Mycobacterium lepraemurium was cultivated on Ogawa egg-yolk medium and its energy coupling mechanisms were investigated. Cell-free extracts prepared from in vitro-grown cells catalyzed phosphorylation coupled to the oxidation of generated NADH, added NADH, and succinate-yielding ratios of phosphorus moles incorporated into high-energy bonds to oxygen atoms utilized (P/O ratios) of 0.75, 0.52, and 0.36, respectively. Ascorbate oxidation alone or in the presence of tetramethyl-p-phenyline-diamine (TMPD) did not yield any adenosine triphosphate (ATP). However, ascorbate in the presence of added cytochrome c was coupled to ATP synthesis and yielded a P/O ratio of 0.12. The oxidative phosphorylation was uncoupled by all of the uncouplers used without any inhibition of oxygen consumption. ATP generation coupled to NADH oxidation was completely inhibited by the flavoprotein inhibitors, such as rotenone and amytal; these inhibitors had no effect, however, on ATP synthesis associated with succinate oxidation. Antimycin A or 2-n-heptyl-4-hydroxy-quinoline-N-oxide (HQNO) and cyanide inhibited markedly the oxidations of NADH and succinate as well as the coupled ATP generation. The phosphorylation coupled to ascorbate plus cytochrome c was not affected by either of the flavoprotein inhibitors or by antimycin A or HQNO, but was completely inhibited by cyanide. The thiol-bearing agents p-chloromercuribenzoate (PCMB) and N-ethylmaleimide were the potent inhibitors of the phosphorylation associated with the oxidation of NADH and succinate. The results indicate that the three energy-coupling sites are functional in the respiratory chain of in vitro-grown M. lepraemurium.
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PMID:Energy generation mechanisms in the in vitro-grown Mycobacterium lepraemurium. 131 45

Fatty acid synthase was purified from Mycobacterium tuberculosis var. bovis BCG. The method developed gave a 23% yield of the synthase and also yielded purified mycocerosic acid synthase. The fatty acid synthase is of unusually large size and composed of two 500-kDa monomers. The amino acid composition of the two synthases was not identical; the N-terminus of the fatty acid synthase was blocked, whereas that of the mycocerosic acid synthase was not. Western blot analysis of crude mycobacterial extracts with polyclonal antibodies prepared against each synthase showed a single band in each case with no cross-reactivity with the other synthase. Fatty acid synthase required both NADH (Km, 11 microM) and NADPH (Km, 14 microM). The Km for acetyl-CoA and malonyl-CoA were 5 and 6 microM, respectively. Fatty acids were released from the synthase as CoA esters. A bimodal distribution of fatty acids was obtained at around C16 and C26. The primer utilization also reflects the de novo synthesis and elongation capabilities of the enzyme; acetyl-CoA was the preferred primer but CoA esters up to C8 but not C12 and C14 could serve as primers, whereas C16 was readily used as a primer for elongation. Addition of CoA and CoA ester-binding oligosaccharides caused enhanced release of C16. Since this mycobacterial fatty acid synthase is twice as large as other multifunctional fatty acid synthases, it is tempting to suggest that this synthase represents a head to tail fusion of two fatty acid synthase genes coding for a double size protein with one-half producing C16 acid and the other elongating the C16 acid to a C26 acid. The monomer of fatty acid synthase from M. smegmatis was immunologically similar and equal in size to the synthase from M. tuberculosis.
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PMID:Purification and characterization of an unusually large fatty acid synthase from Mycobacterium tuberculosis var. bovis BCG. 158 61


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