Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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The NADP-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans (abbreviated Sm-ALDH) belongs to the aldehyde dehydrogenase (ALDH) family. Its catalytic mechanism proceeds via two steps, acylation and deacylation. Its high catalytic efficiency at neutral pH implies prerequisites relative to the chemical mechanism. First, the catalytic Cys284 should be accessible and in a thiolate form at physiological pH to attack efficiently the aldehydic group of the glyceraldehyde-3-phosphate (G3P). Second, the hydride transfer from the hemithioacetal intermediate toward the nicotinamide ring of NADP should be efficient. Third, the nucleophilic character of the water molecule involved in the deacylation should be strongly increased. Moreover, the different complexes formed during the catalytic process should be stabilised. The crystal structures presented here (an apoenzyme named Apo2 with two sulphate ions bound to the catalytic site, the C284S mutant holoenzyme and the ternary complex composed of the C284S holoenzyme and G3P) together with biochemical results and previously published apo and holo crystal structures (named Apo1 and Holo1, respectively) contribute to the understanding of the ALDH catalytic mechanism. Comparison of Apo1 and Holo1 crystal structures shows a Cys284 side-chain rotation of 110 degrees, upon cofactor binding, which is probably responsible for its pK(a) decrease. In the Apo2 structure, an oxygen atom of a sulphate anion interacts by hydrogen bonds with the NH2 group of a conserved asparagine residue (Asn154 in Sm-ALDH) and the Cys284 NH group. In the ternary complex, the oxygen atom of the aldehydic carbonyl group of the substrate interacts with the Ser284 NH group and the Asn154 NH2 group. A substrate isotope effect on acylation is observed for both the wild-type and the N154A and N154T mutants. The rate of the acylation step strongly decreases for the mutants and becomes limiting. All these results suggest the involvement of Asn154 in an oxyanion hole in order to stabilise the tetrahedral intermediate and likely the other intermediates of the reaction. In the ternary complex, the cofactor conformation is shifted in comparison with its conformation in the C284S holoenzyme structure, likely resulting from its peculiar binding mode to the Rossmann fold (i.e. non-perpendicular to the plane of the beta-sheet). This change is likely favoured by a characteristic loop of the Rossmann fold, longer in ALDHs than in other dehydrogenases, whose orientation could be constrained by a conserved proline residue. In the ternary and C284S holenzyme structures, as well as in the Apo2 structure, the Glu250 side-chain is situated less than 4 A from Cys284 or Ser284 instead of 7 A in the crystal structure of the wild-type holoenzyme. It is now positioned in a hydrophobic environment. This supports the pK(a) assignment of 7.6 to Glu250 as recently proposed from enzymatic studies.
J Mol Biol 2000 Jun 30
PMID:Structural and biochemical investigations of the catalytic mechanism of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans. 1086 5

A(1) adenosine receptors (A(1)Rs) are G protein-coupled heptaspanning receptors that interact at the outer face of the plasma membrane with cell surface ecto-adenosine deaminase (ecto-ADA). By affinity chromatography the heat shock cognate protein hsc73 was identified as a cytosolic component able to interact with the third intracellular loop of the receptor. As demonstrated by surface plasmon resonance, purified A(1)Rs interact specifically with hsc73 with a dissociation constant in the nanomolar range (0.5 +/- 0.1 nM). The interaction between hsc73 and A(1)R led to a marked reduction in the binding of the ligands and prevented activation of G proteins, as deduced from (35)S-labeled guanosine-5'-O-(3-thio)triphosphate binding assays. Interestingly this effect was stronger than that exerted by guanine nucleotide analogs, which uncouple receptors from G proteins, and was completely prevented by ADA. As assessed by immunoprecipitation a high percentage of A(1)Rs in cell lysates are coupled to hsc73. A relatively high level of colocalization between A(1)R and hsc73 was detected in DDT(1)MF-2 cells by means of confocal microscopy, and no similar results were obtained for other G protein-coupled receptors. Colocalization between hsc73 and A(1)R was detected in specific regions of rat cerebellum and in the body of cortical neurons but not in dendrites or synapses. Remarkably, agonist-induced receptor internalization leads to the endocytosis of A(1)Rs by two qualitatively different vesicle types, one in which A(1)R and hsc73 colocalize and another in which hsc73 is absent. These results open the interesting possibility that signaling via G protein-coupled receptors may be regulated by heat shock proteins.
Mol Cell Biol 2000 Jul
PMID:The heat shock cognate protein hsc73 assembles with A(1) adenosine receptors to form functional modules in the cell membrane. 1086 72

4-Hydroxy-2-nonenal (HNE) is a highly reactive lipid aldehyde byproduct of the peroxidation of cellular membranes. The structure of HNE features three functional groups, a C1 aldehyde, a C2==C3 double bond, and a C4- hydroxyl group, each of which may contribute to the toxicity of the compound. In addition, the length of the aliphatic chain may influence toxic potency by altering lipophilicity. Using analogous compounds that lacked one or more of the structural moieties, the role of each of these structural motifs in the cytotoxicity of HNE was examined in a mouse alveolar macrophage cell line (RAW 264.7) by a cell survival and growth assay. The importance of these functional groups in the potency of HNE for induction of apoptosis was also examined. The rank order of effects on toxicity was C1---aldehyde >/= C2==C3 double bond >> C4---hydroxyl, with parallel results in both the survival/growth inhibition and apoptosis induction assays. The chain length also influenced toxicity in a series of alpha,beta-unsaturated alkenyl aldehydes, with increasing chain length yielding increasing toxicity. To confirm the importance of the aldehyde moiety, and to examine the role of metabolic detoxification in cellular defenses against HNE toxicity, a RAW 264.7 cell line overexpressing human aldehyde dehydrogenase-3 (hALDH3) was generated. This cell line exhibited nearly complete protection against HNE-protein adduct formation as well as HNE-induced apoptosis. These results illustrate the comparative significance of key structural features of HNE in relation to its potent toxicity and induction of apoptosis.
Mol Pharmacol 2000 Oct
PMID:Structure-activity relationships for growth inhibition and induction of apoptosis by 4-hydroxy-2-nonenal in raw 264.7 cells. 1099 49

There has been considerable recent debate concerning the distances over which levels of allelic association useful for genomic quantitative trait locus (QTL) scans can be detected. We have examined simple sequence repeat (SSR) polymorphisms and two single nucleotide polymorphisms (SNPs) in the region flanking the aldehyde dehydrogenase 2 locus, ALDH2, in populations of Japanese alcoholics and controls. These groups differ significantly in the allele frequencies for the functional SNP in exon XII of this gene located on chromosome 12. The results obtained with SSR markers complement recent investigations with SNPs over similar distances at the TCR alpha/delta locus. Significant allelic association with this marker could be detected for SSRs over distances up to 400 kb and over 37 kb for the SNP thereby extending the distance over which LD at this locus could be detected by an order of magnitude. Furthermore, as a proof of principle, we show that comparisons of allele frequency differences for the SSR markers in the case (alcoholics) and control populations would have detected the ALDH2 marker as a putative QTL. Extending the tests to include alleles at two or three flanking loci suggests that the power to detect QTLs through association can be enhanced significantly.
Hum Mol Genet 2000 Dec 12
PMID:Allele association studies with SSR and SNP markers at known physical distances within a 1 Mb region embracing the ALDH2 locus in the Japanese, demonstrates linkage disequilibrium extending up to 400 kb. 1111 43

Disulfiram, an aldehyde dehydrogenase inhibitor, interferes with normal song maturation when applied to brain nucleus HVC of male zebra finches. We present here evidence from Western blots and enzymatic assays showing that known disulfiram targets other than retinaldehyde-specific aldehyde dehydrogenase (zRalDH) are absent in HVC. These findings are consistent with the conclusion that disulfiram disrupts song maturation by interfering with retinoic acid production.
Brain Res Mol Brain Res 2001 Mar 05
PMID:Molecular targets of disulfiram action on song maturation in zebra finches. 1124 27

Cladosporiumfulvum is a mitosporic ascomycete pathogen of tomato. A study of fungal genes expressed during carbon starvation in vitro identified several genes that were up regulated during growth in planta. These included genes predicted to encode acetaldehyde dehydrogenase (Aldh1) and alcohol oxidase (Aox1). An Aldh1 deletion mutant was constructed. This mutant lacked all detectable ALDH activity, had lost the ability to grow with ethanol as a carbon source, but was unaffected in pathogenicity. Aox1 expression was induced by carbon starvation and during the later stages of infection. The alcohol oxidase enzyme activity has broadly similar properties (Km values, substrate specificity, pH, and heat stability) to yeast enzymes. Antibodies raised to Hansenula polymorpha alcohol oxidase (AOX) detected antigens in Western blots of starved C. fulvum mycelium and infected plant material. Antigen reacting with the antibodies was localized to organelles resembling peroxisomes in starved mycelium and infected plants. Disruption mutants of Aox1 lacked detectable AOX activity and had markedly reduced pathogenicity as assayed by two different measures of fungal growth. These results identify alcohol oxidase as a novel pathogenicity factor and are discussed in relation to peroxisomal metabolism of fungal pathogens during growth in planta.
Mol Plant Microbe Interact 2001 Mar
PMID:Alcohol oxidase is a novel pathogenicity factor for Cladosporium fulvum, but aldehyde dehydrogenase is dispensable. 1127 34

Glycinebetaine is an important osmoprotectant in bacteria, plants, and animals, but only little information is available on the synthesis of glycinebetaine in tree plants. Among four mangrove species, glycinebetaine could be detected only in Avicennia marina. Pinitol was the main osmoprotectant in the other three species. The level of glycinebetaine in A. marina increased under high salinity. Betaine-aldehyde dehydrogenase (BADH) was detected in all four species, but choline monooxygenase could not be detected. A cDNA library was constructed from the leaves of A. marina. Two kinds of BADH cDNAs were isolated, one homologous to the spinach chloroplast BADH, and the other with unique residues SKL at the end of C-terminus. The BADH transcription levels of the former were higher than those of the latter. The levels of the former BADH increased at high salinity whereas those of the latter were independent of salinity. BADHs were expressed in Escherichia coli and purified. Two kinds of A. marina BADHs exhibited similar kinetic and stability properties, but were significantly different from those of spinach BADH. A. marina BADHs efficiently catalyzed the oxidation of betainealdehyde, but not the oxidation of omega-aminoaldehydes and were more stable at high temperature than the spinach BADH.
Plant Mol Biol 2001 Feb
PMID:Molecular cloning and functional characterization of two kinds of betaine-aldehyde dehydrogenase in betaine-accumulating mangrove Avicennia marina (Forsk.) Vierh. 1129 80

This article reviews our knowledge of the ethanol utilization pathway (alc system) in the hyphal fungus Aspergillus nidulans. We discuss the progress made over the past decade in elucidating the two regulatory circuits controlling ethanol catabolism at the level of transcription, specific induction, and carbon catabolite repression, and show how their interplay modulates the utilization of nutrient carbon sources. The mechanisms featuring in this regulation are presented and their modes of action are discussed: First, AlcR, the transcriptional activator, which demonstrates quite remarkable structural features and an original mode of action; second, the physiological inducer acetaldehyde, whose intracellular accumulation induces the alc genes and thereby a catabolic flux while avoiding intoxification; third, CreA, the transcriptional repressor mediating carbon catabolite repression in A. nidulans, which acts in different ways on the various alc genes; Fourth, the promoters of the structural genes for alcohol dehydrogenase (alcA) and aldehyde dehydrogenase (aldA) and the regulatory alcR gene, which exhibit exceptional strength compared to other genes of the respective classes. alc gene expression depends on the number and localization of regulatory cis-acting elements and on the particular interaction between the two regulator proteins, AlcR and CreA, binding to them. All these characteristics make the ethanol regulon a suitable system for induced expression of heterologous protein in filamentous fungi.
Prog Nucleic Acid Res Mol Biol 2001
PMID:Ethanol catabolism in Aspergillus nidulans: a model system for studying gene regulation. 1155 Jul 94

GCN5 is a histone acetyltransferase (HAT) originally identified in Saccharomyces cerevisiae and required for transcription of specific genes within chromatin as part of the SAGA (SPT-ADA-GCN5 acetylase) coactivator complex. Mammalian cells have two distinct GCN5 homologs (PCAF and GCN5L) that have been found in three different SAGA-like complexes (PCAF complex, TFTC [TATA-binding-protein-free TAF(II)-containing complex], and STAGA [SPT3-TAF(II)31-GCN5L acetylase]). The composition and roles of these mammalian HAT complexes are still poorly characterized. Here, we present the purification and characterization of the human STAGA complex. We show that STAGA contains homologs of most yeast SAGA components, including two novel human proteins with histone-like folds and sequence relationships to yeast SPT7 and ADA1. Furthermore, we demonstrate that STAGA has acetyl coenzyme A-dependent transcriptional coactivator functions from a chromatin-assembled template in vitro and associates in HeLa cells with spliceosome-associated protein 130 (SAP130) and DDB1, two structurally related proteins. SAP130 is a component of the splicing factor SF3b that associates with U2 snRNP and is recruited to prespliceosomal complexes. DDB1 (p127) is a UV-damaged-DNA-binding protein that is involved, as part of a complex with DDB2 (p48), in nucleotide excision repair and the hereditary disease xeroderma pigmentosum. Our results thus suggest cellular roles of STAGA in chromatin modification, transcription, and transcription-coupled processes through direct physical interactions with sequence-specific transcription activators and with components of the splicing and DNA repair machineries.
Mol Cell Biol 2001 Oct
PMID:Human STAGA complex is a chromatin-acetylating transcription coactivator that interacts with pre-mRNA splicing and DNA damage-binding factors in vivo. 1156 63

Cell proliferation requires precise control to prevent mutations from replication of (unrepaired) damaged DNA in cells exposed spontaneously to mutagens. Here we show that the modified human DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (R-MGMT), formed from the suicidal repair of the mutagenic O(6)-alkylguanine (6RG) lesions by MGMT in the cells exposed to alkylating carcinogens, functions in such control by preventing the estrogen receptor (ER) from transcription activation that mediates cell proliferation. This function is in contrast to the phosphotriester repair domain of bacterial ADA protein, which acts merely as a transcription activator for its own synthesis upon repair of phosphotriester lesions. First, MGMT, which is constitutively present at active transcription sites, coprecipitates with the transcription integrator CREB-binding protein CBP/p300 but not R-MGMT. Second, R-MGMT, which adopts an altered conformation, utilizes its exposed VLWKLLKVV peptide domain (codons 98 to 106) to bind ER. This binding blocks ER from association with the LXXLL motif of its coactivator, steroid receptor coactivator-1, and thus represses ER effectively from carrying out transcription that regulates cell growth. Thus, through a change in conformation upon repair of the 6RG lesion, MGMT switches from a DNA repair factor to a transcription regulator (R-MGMT), enabling the cell to sense as well as respond to mutagens. These results have implications in chemotherapy and provide insights into the mechanisms for linking transcription suppression with transcription-coupled DNA repair.
Mol Cell Biol 2001 Oct
PMID:The modified human DNA repair enzyme O(6)-methylguanine-DNA methyltransferase is a negative regulator of estrogen receptor-mediated transcription upon alkylation DNA damage. 1156 93


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