Functional studies of aldo-keto reductases in Saccharomyces cerevisiae

We utilized the budding yeast Saccharomyces cerevisiae as a model to systematically explore physiological roles for yeast and mammalian aldo-keto reductases. Six open reading frames encoding putative aldo-keto reductases were identified when the yeast genome was queried against the sequence for human aldose reductase, the prototypical mammalian aldo-keto reductase. Recombinant proteins produced from five of these yeast open reading frames demonstrated NADPH-dependent reductase activity with a variety of aldehyde and ketone substrates. A triple aldo-keto reductase null mutant strain demonstrated a glucose-dependent heat shock phenotype which could be rescued by ectopic expression of human aldose reductase. Catalytically-inactive mutants of human or yeast aldo-keto reductases failed to effect a rescue of the heat shock phenotype, suggesting that the phenotype results from either an accumulation of one or more unmetabolized aldo-keto reductase substrates or a synthetic deficiency of aldo-keto reductase products generated in response to heat shock stress. These results suggest that multiple aldo-keto reductases fulfill functionally redundant roles in the stress response in yeast.     

Hepatocyte susceptibility to glyoxal is dependent on cell thiamin content

Glyoxal, a reactive dicarbonyl, is detoxified primarily by the glyoxalase system utilizing glutathione (GSH) and by the aldo-keto reductase enzymes which utilizes NAD[P]H as the co-factor. Thiamin (Vitamin B(1)) is an essential coenzyme for transketolase (TK) that is part of the pentose phosphate pathway which helps maintain cellular NADPH levels. NADPH plays an intracellular role in regenerating glutathione (GSH) from oxidized GSH (GSSG), thereby increasing the antioxidant defenses of the cell. In this study we have focused on the prevention of glyoxal toxicity by supplementation with thiamin (3mM). Thiamin was cytoprotective and restored NADPH levels, glyoxal detoxification and mitochondrial membrane potential. Hepatocyte reactive oxygen species (ROS) formation, lipid peroxidation and GSH oxidation were decreased. Furthermore, hepatocytes were made thiamin deficient with oxythiamin (3mM) as measured by the decreased hepatocyte TK activity. Under thiamin deficient conditions a non-toxic dose of glyoxal (2mM) became cytotoxic and glyoxal metabolism decreased; while ROS formation, lipid peroxidation and GSH oxidation was increased.     

Identification of catalytic residues in glyoxal oxidase by targeted mutagenesis

Glyoxal oxidase is a copper metalloenzyme produced by the wood-rot fungus Phanerochaete chrysosporium as an essential component of its extracellular lignin degradation pathways. Previous spectroscopic studies on glyoxal oxidase have demonstrated that it contains a free radical-coupled copper active site remarkably similar to that found in another fungal metalloenzyme, galactose oxidase. Alignment of primary structures has allowed four catalytic residues of glyoxal oxidase to be targeted for site-directed mutagenesis in the recombinant protein. Three glyoxal oxidase mutants have been heterologously expressed in both a filamentous fungus (Aspergillus nidulans) and in a methylotrophic yeast (Pichia pastoris), the latter expression system producing as much as 2 g of protein per liter of culture medium under conditions of high density methanol-induced fermentation. Biochemical and spectroscopic characterization of the mutant enzymes supports structural correlations between galactose oxidase and glyoxal oxidase, clearly identifying the catalytically important residues in glyoxal oxidase and demonstrating the functions of each of these residues.     

米曲霉木糖还原酶基因的克隆及序列分析

木糖还原酶（XR, EC 1.1.1.21）是真菌微生物代谢木糖的关键酶之一。本文以米曲霉基因组DNA为模板,克隆木糖还原酶基因（xr,GenBank登录号：FJ957890.1）,并对XR的序列、系统进化树、理化性质及蛋白结构等进行生物信息学分析。结果表明： xr基因序列长1449 bp,其中开放阅读框长960 bp,编码319个氨基酸,蛋白质分子质量35.9 kDa,等电点为5.78;米曲霉XR与其他菌种XR有较高的同一性,含有醛酮还原酶家族的两个指纹结构和一个参与辅酶结合活性位点指纹结构,以及醛酮还原酶家族典型的（β/α）8 TIM桶结构,说明米曲霉XR属于醛酮还原酶家族。     

Conversion of methylglyoxal to acetol by Escherichia coli aldo-keto reductases

Methylglyoxal (MG) is a toxic metabolite known to accumulate in various cell types. We detected in vivo conversion of MG to acetol in MG-accumulating Escherichia coli cells by use of (1)H nuclear magnetic resonance ((1)H-NMR) spectroscopy. A search for homologs of the mammalian aldo-keto reductases (AKRs), which are known to exhibit activity to MG, revealed nine open reading frames from the E. coli genome. Based on both sequence similarities and preliminary characterization with (1)H-NMR for crude extracts of the corresponding mutant strains, we chose five genes, yafB, yqhE, yeaE, yghZ, and yajO, for further study. Quantitative assessment of the metabolites produced in vitro from the crude extracts of these mutants and biochemical study with purified AKRs indicated that the yafB, yqhE, yeaE, and yghZ genes are involved in the conversion of MG to acetol in the presence of NADPH. When we assessed their in vivo catalytic activities by creating double mutants, all of these genes except for yqhE exhibited further sensitivities to MG in a glyoxalase-deficient strain. The results imply that the glutathione-independent detoxification of MG can occur through multiple pathways, consisting of yafB, yqhE, yeaE, and yghZ genes, leading to the generation of acetol.     

Amplification of the ribonucleotide reductase small subunit gene: analysis of novel joints and the mechanism of gene duplication in vaccinia virus.

Amplification of the M2 gene encoding the small subunit of ribonucleotide reductase (EC 1.17.4.1) was analyzed in a collection of vaccinia virus (VV) isolates selected for resistance to 5 mM hydroxyurea (HU). Most of the mutants harbored tandem direct repeat arrays of the M2 gene, but several had duplicated M2 as an inverted repeat by genomic rearrangements involving the chromosomal termini. Novel joints formed by direct repeats were mapped, amplified in vitro, and sequenced. The junctions were simple fusions between DNA downstream and upstream of the M2 gene. Lack of sequence homology at the breakpoints indicated that the initial genomic rearrangements leading to gene amplification were due to nonhomologous recombination events.     

Analysis of flavanone 3-hydroxylase in Arabidopsis seedlings. Coordinate regulation with chalcone synthase and chalcone isomerase.

A genomic clone encoding flavanone 3-hydroxylase (F3H) was isolated from Arabidopsis thaliana. The deduced amino acid sequence is 72 to 94% identical to all previously reported F3H proteins. Low-stringency DNA blot analysis indicated that F3H is encoded by a single gene in Arabidopsis. The F3H locus was mapped to the bottom of chromosome 3 and therefore does not correspond to any of the 13 flavonoid-deficient transparent testa mutants for which a map position is known. Analysis of gene expression in etiolated seedlings exposed to white light and in two putative regulatory mutants, ttg and tt8, demonstrated that the Arabidopsis F3H gene is coordinately expressed with chalcone synthase and chalcone isomerases is seedlings, whereas dihydroflavonol reductase expression is controlled by distinct regulatory mechanisms. The F3H gene may represent a pivotal point in the regulation of flavonoid biosynthesis because its expression is coordinated with different subsets of genes in different plant species.     

Two-step oxidation of glycerol to glyceric acid catalyzed by the Phanerochaete chrysosporium glyoxal oxidase

Glyoxal oxidase of P. chrysosporium is a radical copper oxidase that catalyzes oxidation of aldehydes to carboxylic acids coupled to dioxygen reduction to H(2)O(2). In addition to known substrates, glycerol is also found to be a substrate for glyoxal oxidase. During enzyme turnover, glyoxal oxidase undergoes a reversible inactivation, probably caused by loss of the active site free radical, resulting in short-lasting enzyme activities and undetectable substrate conversions. Enzyme activity could be extended by including two additional enzymes, horseradish peroxidase and catalase, in addition to a redox chemical activator, such as Mn(III) (or Mn(II)+H(2)O(2)) or hexachloroiridate. Using this three-enzyme system glycerol was converted in glyceric acid in a two-step reaction, with glyceraldehyde as intermediate. A possible operation mechanism is proposed in which the three enzymes would work coordinately allowing to maintain a sustained glyoxal oxidase activity. In the course of its catalytic cycle, glyoxal oxidase alternates between two functional and interconvertible reduced and oxidized forms resulting from a two-electron transfer process. However, glyoxal oxidase can also undergo an one-electron reduction to a catalytically inactive form lacking the active site free radical. Horseradish peroxidase could use glyoxal oxidase-generated H(2)O(2) to oxidize Mn(II) to Mn(III) which, in turn, would reoxidize and reactivate the inactive form of glyoxal oxidase. Catalase would remove the excess of H(2)O(2) generated during the reaction. In spite of the improvement achieved using the three-enzyme system, glyoxal oxidase inactivation still occurred, which resulted in low substrate conversions. Possible causes of inactivation, including end-product inhibition, are discussed.     

The structural organization of the human aldehyde reductase gene, AKR1A1, and mapping to chromosome 1p33-->p32.

Genomic DNA encoding for human aldehyde reductase (AKR1A1), a member of the aldo-keto reductase superfamily, was isolated and characterized. The genomic DNA is approximately 16 kb in length and contains eight exons which encode the entire coding region and the 3'-untranslated sequences. AKR1A1 was localized on chromosome 1p33-->p32 by fluorescence in situ hybridization.     

Cloning and characterization of a NAD+-dependent glycerol-3-phosphate dehydrogenase gene from Candida glycerinogenes, an industrial glycerol producer.

The osmotolerant yeast Candida glycerinogenes produces glycerol as a major metabolite on an industrial scale, but the underlying molecular mechanisms are poorly understood. We cloned and characterized a 4900-bp genomic fragment containing the CgGPD gene encoding a glycerol-3-phosphate dehydrogenase homologous to GPD genes in other yeasts using degenerate primers in conjunction with inverse PCR. Sequence analysis revealed a 1167-bp open reading frame encoding a putative peptide of 388 deduced amino acids with a molecular mass of 42 695 Da. The CgGPD gene consisted of an N-terminal NAD(+)-binding domain and a central catalytic domain, whereas seven stress response elements were found in the upstream region. Functional analysis revealed that Saccharomyces cerevisiae gpd1Delta and gpd1Delta/gpd2Delta osmosensitive mutants transformed with CgGPD were restored to the wild-type phenotype when cultured in high osmolarity media, suggesting that it is a functional GPD protein. Transformants also accumulated glycerol intracellularly and GPD-specific activity increased significantly when stressed with NaCl, whereas the S. cerevisiae mutants transformed with the empty plasmid showed only slight increases. The full-length CgGPD gene sequence including upstream and downstream regions has been deposited in GenBank under accession no. EU186536.     

Molecular characterization of LbpB, the second lactoferrin-binding protein of Neisseria meningitidis

The lbpA gene of Neisseria meningitidis encodes an outer membrane lactoferrin-binding protein and shows homology to the transferrin-binding protein, TbpA. Previously, we have detected part of an open reading frame upstream of lbpA . The putative product of this open reading frame, tentatively designated lbpB showed homology to the transferrin-binding protein TbpB, suggesting that the lactoferrrin receptor, like the transferrin receptor, consists of two proteins. The complete nucleotide sequence of lbpB was determined. The gene encodes a 77.5 kDa protein, probably a lipoprotein, with homology, 33% identity to the TbpB of N . meningitidis . A unique feature of LbpB is the presence of two stretches of negatively charged residues, which might be involved in lactoferrin binding. Antisera were raised against synthetic peptides corresponding to the C-terminal part of the putative protein and used to demonstrate that the gene is indeed expressed. Consistent with the presence of a putative Fur binding site upstream of the lbpB gene, expression of both LbpA and LbpB was proved to be iron regulated in Western blot experiments. The LbpB protein appeared to be less stable than TbpB in SDS-containing sample buffer. Isogenic mutants lacking either LbpA or LbpB exhibited a reduced ability to bind lactoferrin. In contrast to the lbpB mutant, the lbpA mutant was completely unable to use lactoferrin as a sole source of iron.     

Insensitivity of cardiac phosphofructokinase to adrenergic activation in Zucker rats. A post-receptor defect.

Glyoxal bis(guanylhydrazone), the parent compound of methylglyoxal bis(guanylhydrazone), was synthesized and tested for its ability to inhibit the biosynthesis of polyamines. It was found to be a powerful competitive inhibitor of adenosylmethionine decarboxylase (EC 4.1.1.50), yet the lack of the methyl group at the glyoxal portion increased the apparent Ki value for the enzyme by about 30-fold in comparison with methylglyoxal bis(guanylhydrazone). Glyoxal bis(guanylhydrazone) inhibited diamine oxidase (EC 1.4.3.6) activity as effectively as did methylglyoxal bis(guanylhydrazone). The cellular accumulation curves of glyoxal bis(guanylhydrazone) in L1210 cells were practically superimposable with those of methylglyoxal bis(guanylhydrazone), and the uptake of both compounds was distinctly stimulated by a prior treatment with 2-difluoromethylornithine. The drug decreased the concentration of spermidine in a dose-dependent manner and, in contrast with methylglyoxal bis(guanylhydrazone), without a concomitant accumulation of putrescine. The fact that putrescine concentrations were decreased in cells exposed to glyoxal bis(guanylhydrazone) was, at least in part, attributable to an inhibition of ornithine decarboxylase (EC 4.1.1.17) activity in cells treated with the compound. Under these experimental conditions equivalent concentrations of methylglyoxal bis(guanylhydrazone) [1,1'-[(methylethanediylidine)dinitrilo]diguanidine] elicited large increases in the enzyme activity. When combined with difluoromethylornithine, glyoxal bis(guanylhydrazone) potentiated the growth-inhibitory effect of that drug. Taking into consideration the proven anti-leukaemic activity of glyoxal bis(guanylhydrazone), its effectiveness to inhibit spermidine biosynthesis (without raising the concentration of putrescine) as well as its suitability for combined use with inhibitors of ornithine decarboxylase, this drug is apparently worthy of further testing in tumour-bearing animals, especially in combination with difluoromethylornithine or related inhibitors of ornithine decarboxylase.