Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The crystal structure of malate dehydrogenase from Escherichia coli has been determined with a resulting R-factor of 0.187 for X-ray data from 8.0 to 1.87 A. Molecular replacement, using the partially refined structure of porcine mitochondrial malate dehydrogenase as a probe, provided initial phases. The structure of this prokaryotic enzyme is closely homologous with the mitochondrial enzyme but somewhat less similar to cytosolic malate dehydrogenase from eukaryotes. However, all three enzymes are dimeric and form the subunit-subunit interface through similar surface regions. A citrate ion, found in the active site, helps define the residues involved in substrate binding and catalysis. Two arginine residues, R81 and R153, interacting with the citrate are believed to confer substrate specificity. The hydroxyl of the citrate is hydrogen-bonded to a histidine, H177, and similar interactions could be assigned to a bound malate or oxaloacetate. Histidine 177 is also hydrogen-bonded to an aspartate, D150, to form a classic His.Asp pair. Studies of the active site cavity indicate that the bound citrate would occupy part of the site needed for the coenzyme. In a model building study, the cofactor, NAD, was placed into the coenzyme site which exists when the citrate was converted to malate and crystallographic water molecules removed. This hypothetical model of a ternary complex was energy minimized for comparison with the structure of the binary complex of porcine cytosolic malate dehydrogenase. Many residues involved in cofactor binding in the minimized E. coli malate dehydrogenase structure are homologous to coenzyme binding residues in cytosolic malate dehydrogenase. In the energy minimized structure of the ternary complex, the C-4 atom of NAD is in van der Waals' contact with the C-3 atom of the malate. A catalytic cycle involves hydride transfer between these two atoms.
J Mol Biol 1992 Aug 05
PMID:Crystal structure of Escherichia coli malate dehydrogenase. A complex of the apoenzyme and citrate at 1.87 A resolution. 150 30

Citrate is fermented by Klebsiella pneumoniae to 2 acetate, 0.5 formate and 1.2 CO2. The formation of less than 1 formate and greater than 1 CO2 per citrate can be accounted for by the oxidation of formate to CO2 in order to provide reducing equivalents for the assimilation of citrate into cell carbon. A membrane-bound electron transport chain is apparently involved in NADH synthesis by these cells. The electrons from formate oxidation to CO2 are used to reduce ubiquinone to ubiquinol by membrane-bound formate dehydrogenase and ubiquinol further delivers its electrons to NAD+, if this endergonic reaction is powered by delta mu Na+. The endogenous NADH level of K. pneumoniae cells thus increased in the presence of formate in response to a delta pNa+ greater than -100 mV. NADH formation was completely abolished in the presence of oxygen or after addition of hydroxyquinoline-N-oxide, a specific inhibitor of the Na(+)-translocating NADH:ubiquinone oxidoreductase. The increase of endogenous NADH was dependent on the delta pNa+ applied to the cells. Inverted membrane vesicles of K. pneumoniae catalysed the reduction of NAD+ to NADH with formate as electron donor after application of delta mu Na+ of about 120 mV consisting of delta pNa+ of 60 mV and delta psi of the same magnitude. Neither the delta pNa+ nor the delta psi of this size alone was sufficient to drive the endergonic reaction. Strictly anaerobic conditions were required for NADH formation and hydroxyquinoline-N-oxide completely inactivated the reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Microbiol 1992 Jul
PMID:NADH formation by Na(+)-coupled reversed electron transfer in Klebsiella pneumoniae. 150 43

The alcohol dehydrogenase (ADHase) enzyme catalyses the oxidation of alcohols to aldehydes or ketones using NAD+ as a cofactor. Functional ADHase from Drosophila lebanonensis is a dimer, with a monomeric molecular weight of 27,000 and with 254 residues in each polypeptide chain. Crystals of the protein have been grown with and without NAD+. Two crystal forms have been observed. Most crystals are plate-like, 0.05 mm in their shortest dimension and up to 0.4 mm in their longest dimension. These crystals are generally too small to diffract efficiently using conventional X-ray sources, so preliminary studies were carried out using the Synchrotron Radiation Source at the SERC Daresbury Laboratory. Twinning was a severe problem with this crystal form. The second form is grown in the absence of NAD+ but with DL-dithiothreitol present. These crystals grow more evenly and diffract to better than 2 A resolution. They are monoclinic, with cell dimensions, a = 81.24(6) A, b = 55.75(4) A, c = 109.60(7) A and beta = 94.26(9) degrees, space group P2(1). There are two dimers in the asymmetric unit, but at low resolution a rotated cell with one dimer per asymmetric unit can be obtained.
J Mol Biol 1992 Sep 05
PMID:Preliminary X-ray crystallographic studies on alcohol dehydrogenase from Drosophila. 152

At 3-4 degrees C, the transport of 3-O-methyl-D-glucose (30 mM) was severely impaired in islets prepared from adult rats injected with streptozotocin during the neonatal period. However, at 37 degrees C, the first and second phase of glucose-stimulated insulin release were decreased to the same relative extent in perifused islets of diabetic, as compared to control, animals. Moreover, the time-related increase in the oxidative response of the islets to 16.7 mM D-glucose was less pronounced in diabetic than control rats. The activity of the mitochondrial FAD-linked glycerophosphate dehydrogenase in islet homogenates of diabetic rats only represented one-fifth of that found in control rats, whereas the activity of the cytosolic NAD-glycerophosphate dehydrogenase was comparable in both types of rats. This coincided with the fact that a rise in D-glucose concentration from 2.8 to 16.7 mM failed to increase significantly L-[2-3H]glycerol conversion to 3HOH in islets from diabetic rats, in contrast to the situation found in control animals. The activity of 2-ketoglutarate dehydrogenase in islet homogenates when expressed per microgram protein was not different in control and diabetic rats. Likewise, the ratio between D-[6-14C]glucose oxidation and D-[3,4-14C]glucose oxidation and the capacity of either a non-metabolized analog of L-leucine or 3-phenylpyruvate to preferentially stimulated D-[6-14C]glucose oxidation relative to D-[5-3H]glucose utilization were both unaffected in islets from diabetic rats. These findings argue against the existence of a primary defect in the Krebs cycle of diabetic rats. It is proposed that, despite an obvious alteration of the hexose transport system in the islet cells of diabetic rats, the preferential impairment of the B-cell secretory response to D-glucose, as distinct from other secretagogues, in this model of non-insulin-dependent diabetes is mainly attributable to the low activity of FAD-linked glycerophosphate dehydrogenase, resulting in a decreased metabolic flow through the glycerol phosphate shuttle and a reduced rate of aerobic glycolysis.
Mol Cell Endocrinol 1992 Feb
PMID:Study of hexose transport, glycerol phosphate shuttle and Krebs cycle in islets of adult rats injected with streptozotocin during the neonatal period. 153 53

A 4 kb SalI fragment from Azospirillum brasilense Sp7 that shares homology with a 6.8 kb EcoRI fragment carrying nodGEFH and part of nodP of Rhizobium meliloti 41 was cloned in pUC18 to yield pAB503. The nucleotide sequence of a 2 kb SalI-SmaI fragment of the pAB503 insert revealed an open reading frame, named ORF3, encoding a polypeptide sharing 40% identity with R. meliloti NodG. The deduced polypeptide also shared 60% identity with the Alcaligenes eutrophus NADPH-dependent acetoacetyl-CoA (AA-CoA) reductase, encoded by the phbB gene and involved in poly-beta-hydroxybutyrate (PHB) synthesis. Northern blot analysis and promoter extension mapping indicated that ORF3 is expressed as a monocistronic operon from a promoter that resembles the Escherichia coli sigma 70 consensus promoter. An ORF3-lacZ translational fusion was constructed and was very poorly expressed in E. coli, but was functional and constitutively expressed in Azospirillum. Tn5-Mob insertions in ORF3 did not affect growth, nitrogen fixation, PHB synthesis or NAD(P)H-linked AA-CoA reductase activity. An ORF3 DNA sequence was used to probe total DNA of several Azospirillum strains. No ORF3 homologues were found in A. irakense, A. amazonense, A. halopraeferens or in several A. lipoferum strains.
Mol Gen Genet 1992 Feb
PMID:Characterization of an Azospirillum brasilense Sp7 gene homologous to Alcaligenes eutrophus phbB and to Rhizobium meliloti nodG. 153 94

Constitutive expression of human nuclear NAD+: protein ADP-ribosyltransferase (polymerizing) [pADPRT; poly(ADP-ribose)polymerase; EC 2.4.2.30] as an active enzyme in Saccharomyces cerevisiae, under the control of the alcohol dehydrogenase promoter, was only possible with simultaneous inhibition of ADP-ribosylation by 3-methoxybenzamide. Induction of fully active pADPRT from the inducible galactose epimerase promoter resulted in inhibition of cell division and morphological changes reminiscent of cell cycle mutants. Expression of a pADPRT cDNA truncated at its 5' end had no influence on cell proliferation at all. Obviously the amino-terminal part of the DNA binding domain containing the first "zinc finger", which is essential for inducibility of pADPRT activity by DNA breaks, is also required for inhibition of cell growth on expression in yeast. Full-length as well as truncated pADPRT molecules were directed to the cell nucleus where the fully active enzyme produced large amounts of poly(ADP-ribose) by automodification. Since pADPRT turned out to be the only target for ADP-ribosylation in these cells, elevated levels of poly(ADP-ribose) were the most likely cause of inhibition of cell division, presumably resulting from interaction with chromosomal proteins.
Mol Gen Genet 1992 Mar
PMID:Inhibition of cell proliferation in Saccharomyces cerevisiae by expression of human NAD+ ADP-ribosyltransferase requires the DNA binding domain ("zinc fingers"). 155 29

Redox interconversion of glutathione reductase was studied in situ with S. cerevisiae. The enzyme was more sensitive to redox inactivation in 24 hour-starved cells than in freshly-grown ones. While 5 microM NADPH or 100 microM NADH caused 50% inactivation in normal cells in 30 min, 0.75 microM NADPH or 50 microM NADH promoted a similar effect in starved cells. GSSG reactivated the enzyme previously inactivated by NADPH, ascertaining that the enzyme was subjected to redox interconversion. Low EDTA concentrations fully protected the enzyme from NADPH inactivation, thus confirming the participation of metals in such a process. Extensive inactivation was obtained in permeabilized cells incubated with glucose-6-phosphate or 6-phosphogluconate, in agreement with the very high specific activities of the corresponding dehydrogenases. Some inactivation was also observed with malate, L-lactate, gluconate or isocitrate in the presence of low NADP+ concentrations. The inactivation of yeast glutathione reductase has also been studied in vivo. The activity decreased to 75% after 2 hours of growth with glucono-delta-lactone as carbon source, while NADPH rose to 144% and NADPH+ fell to 86% of their initial values. Greater changes were observed in the presence of 1.5 microM rotenone: enzymatic activity descended to 23% of the control value, while the NADH/NAD+ and NADPH/NADP+ ratios rose to 171% and 262% of their initial values, respectively. Such results indicate that the lowered redox potential of the pyridine nucleotide pool existing when glucono-delta-lactone is oxidized promotes in vivo inactivation of glutathione reductase.
Mol Cell Biochem 1992 Mar 25
PMID:Glutathione reductase from Saccharomyces cerevisiae undergoes redox interconversion in situ and in vivo. 158 2

Sequences of 47 members of the Zn-containing alcohol dehydrogenase (ADH) family were aligned progressively, and an evolutionary tree with detailed branch order and branch lengths was produced. The alignment shows that only 9 amino acid residues (of 374 in the horse liver ADH sequence) are conserved in this family; these include eight Gly and one Val with structural roles. Three residues that bind the catalytic Zn and modulate its electrostatic environment are conserved in 45 members. Asp 223, which determines specificity for NAD, is found in all but the two NADP-dependent enzymes, which have Gly or Ala. Ser or Thr 48, which makes a hydrogen bond to the substrate, is present in 46 members. The four Cys ligands for the structural zinc are conserved except in zeta-crystallin, the sorbitol dehydrogenases, and two bacterial enzymes. Analysis of the evolutionary tree gives estimates of the times of divergence for different animal ADHs. The human class II (pi) and class III (chi) ADHs probably diverged about 630 million years ago, and the newly identified human ADH6 appeared about 520 million years ago, implying that these classes of enzymes may exist or have existed in all vertebrates. The human class I ADH isoenzymes (alpha, beta, and gamma) diverged about 80 million years ago, suggesting that these isoenzymes may exist or have existed in all primates. Analysis of branch lengths shows that these plant ADHs are more conserved than the animal ones and that class III ADHs are more conserved than class I ADHs. The rate of acceptance of point mutations (PAM units) shows that selection pressure has existed for ADHs, implying that these enzymes play definite metabolic roles.
J Mol Evol 1992 Jun
PMID:Progressive sequence alignment and molecular evolution of the Zn-containing alcohol dehydrogenase family. 159 44

The ability of the rabbit blastocyst to reduce nitroblue tetrazolium (NBT) to formazan in the presence of cyanide was assayed as an indicator of extramitochondrial oxidase activity capable of generating the superoxide radical. A cytochemical method initially developed for the detection and localization of hydrogen peroxide production at the ultrastructural level in phagocytosing leukocytes (Briggs et al.: J Cell Biol 67:566, 1975) was also applied to the blastocyst. The results demonstrate that the rabbit blastocyst acquires the ability to reduce NBT by a cyanide-insensitive process and to generate hydrogen peroxide between the fourth and fifth days postcoitum. The enzymatic activity responsible is apparently an NAD(P)H-dependent oxidase in the outer, microvillous plasma membrane of the trophoblast.
Mol Reprod Dev 1992 Feb
PMID:Cyanide-resistant reduction of nitroblue tetrazolium and hydrogen peroxide production by the rabbit blastocyst. 159 79

Metabolism of 11-deoxycorticosterone (DOC) by hamster adrenal mitochondria gives 19-hydroxy-DOC and corticosterone (via 11-hydroxylation) in approximately equal yields. The ratio of 19- to 11-hydroxylation was invariant with changes in concentration of substrate or a competitive inhibitor. It is most likely, therefore, that a single 11,19-hydroxylase catalyzes both oxidations. Both primary products are further oxidized to the corresponding carbonyl analogs, 19-oxo-DOC and 11-dehydrocorticosterone, at rates that are approx. 20% of their rates of formation. The oxidation of 11-dehydrocorticosterone is catalyzed by a dehydrogenase utilizing either NAD or NADP while the oxidation of 19-hydroxy-DOC is catalyzed by an oxidase requiring NADPH. The 11-dehydrocorticosterone is stable in this enzyme preparation while 19-oxo-DOC is metabolized to two additional products, which are tentatively identified as 19-oic-DOC and 19-norcorticosterone. 19-nor-DOC was found to be hydroxylated at a rate that is 20% faster than the rate for DOC under the same conditions. It is therefore possible that 19-norcorticosterone can arise from 19-oic-DOC via decarboxylation to 19-nor-DOC and subsequent 11-hydroxylation, but the kinetics of its formation suggest that it may actually be formed directly from 19-oxo-DOC without free intermediates. 4-Androstene-3,17-dione and 17-hydroxy-DOC were also substrates for this 11,19-hydroxylase, but 18-hydroxy-DOC was not. Maintenance of hamsters on a low sodium diet had no effect on the metabolism of DOC by the isolated adrenal mitochondria.
J Steroid Biochem Mol Biol 1992 May
PMID:Metabolism of 11-deoxycorticosterone by hamster adrenal mitochondria. 160 48


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