Purification and Properties of NAD-Dependent Sorbitol Dehydrogenase from Apple Fruit

This is the first report of the purification of NAD-dependentsorbitol dehydrogenase (NAD-SDH) from a plant source. The enzymewas extracted from apple (Malus domestica cv. Ourin) fruit andpurified until it appeared as a single polypeptide chain ona gel after SDS-PAGE. From the apparent molecular mass of 62kDa obtained by SDS-PAGE and that of 120 kDa by gel filtration,the enzyme appeared to be a homodimer. Maximum rates of oxidationof sorbitol and reduction of fructose were observed at pH 9.6and pH 6.0, respectively. The K_m for oxidation of sorbitol was40.3 mM and that for reduction of fructose was 215 mM. The maximumrate of oxidation of sorbitol was about 10 times higher thanthat of the reduction of fructose. The results of the kineticanalysis strongly suggest that in vivo the enzyme would favorthe conversion of sorbitol to fructose over the reverse reaction.None of the divalent cations tested had any effect on the oxidationof sorbitol by NAD-SDH. The reaction catalyzed by NAD-SDH wasnot specific to sorbitol and other substrates could also beoxidized. Among the tested substrates, ethyl alcohol had a particularlyhigh affinity for the enzyme. (Received February 2, 1994; Accepted May 31, 1994)     

Shifted Cytosolic NADP+-Dependent Isocitrate Dehydrogenase on 2-D Gel in the Brain of Genetically Epileptic El Mice

We observed a spot on two-dimensional (2-D) gel in the epileptic mutant strain El mice with a similar molecular weight but with a different isoelectric point of approximately 0.2, compared with its mother strain ddY mice. The collected protein from the El mice was identified as cytosolic NADP⁺-dependent isocitrate dehydrogenase by internal amino acid sequencing. The enzyme is known to be maximally active during the development of the brain and to play an important role in NADPH production for fatty acids and cholesterol synthesis. In addition, alterations in cholesterol synthesis early in the development of the mammalian brain have been reported to lead to chronic epilepsy. The results in the present study therefore suggest that cytosolic NADP⁺-dependent isocitrate dehydrogenase might be involved in the epileptogenesis of the El mouse.     

NADP+-Preferring d-Lactate Dehydrogenase from Sporolactobacillus inulinus

Hydroxy acid dehydrogenases, including l- and d-lactate dehydrogenases (L-LDH and D-LDH), are responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids and extensively used in a wide range of biotechnological applications. A common feature of LDHs is their high specificity for NAD⁺ as a cofactor. An LDH that could effectively use NADPH as a coenzyme could be an alternative enzymatic system for regeneration of the oxidized, phosphorylated cofactor. In this study, a d-lactate dehydrogenase from a Sporolactobacillus inulinus strain was found to use both NADH and NADPH with high efficiencies and with a preference for NADPH as its coenzyme, which is different from the coenzyme utilization of all previously reported LDHs. The biochemical properties of the D-LDH enzyme were determined by X-ray crystal structural characterization and in vivo and in vitro enzymatic activity analyses. The residue Asn¹⁷⁴ was demonstrated to be critical for NADPH utilization. Characterization of the biochemical properties of this enzyme will contribute to understanding of the catalytic mechanism and provide referential information for shifting the coenzyme utilization specificity of 2-hydroxyacid dehydrogenases.     

NADP+-Dependent D-Arabinose Dehydrogenase Shows a Limited Contribution to Erythroascorbic Acid Biosynthesis and Oxidative Stress Resistance in Saccharomyces cerevisiae

The molecular aspects and physiological significance of NADP⁺-dependent D-arabinose dehydrogenase (ARA), which is thought to function in the biosynthesis of an analog of ascorbic acid, D-erythroascorbic acid in yeasts, were examined. A large subunit of ARA, Ara1p produced in E. coli, was purified as a homodimer, some of which was degraded at the N-terminus. It showed sufficient ARA activity. Degradation of Ara1p occurs naturally in yeast cells, and the small subunit of ARA previously thought as is, in fact, a naturally occuring degradation product of Ara1p. A deficient mutant of ARA1 lost almost all NADP⁺-ARA activity, but intracellular D-erythroascorbic acid was only halved. This mutant showed increased susceptibility to H₂O₂ and diamide but not to menadione or tert-butylhydroperoxide. Feeding D-arabinose to mutant cells led to increases in intracellular D-erythroascorbic acid, suggesting the presence of another ARA isozyme. The deficient mutant of ARA1 recovered resistance to H₂O₂ with feeding of D-arabinose. Our results suggest that the direct contributions of Ara1p both to D-erythroascorbic acid biosynthesis and to oxidative stress resistance are quite limited.     

Efficient Bioethanol Production by a Recombinant Flocculent Saccharomyces cerevisiae Strain with a Genome-Integrated NADP+-Dependent Xylitol Dehydrogenase Gene

The recombinant industrial Saccharomyces cerevisiae strain MA-R5 was engineered to express NADP⁺-dependent xylitol dehydrogenase using the flocculent yeast strain IR-2, which has high xylulose-fermenting ability, and both xylose consumption and ethanol production remarkably increased. Furthermore, the MA-R5 strain produced the highest ethanol yield (0.48 g/g) from nonsulfuric acid hydrolysate of wood chips.Successful fermentation of lignocellulosic biomass to ethanol is dependent on efficient utilization of d-xylose, which is the second most common fermentable sugar in the hydrolysate. Although the well-known fermentative yeast Saccharomyces cerevisiae is one of the most effective ethanol-producing organisms for hexose sugars, it is not able to ferment d-xylose. However, S. cerevisiae can metabolize an isomerization product of d-xylose, d-xylulose, which is phosphorylated to d-xylulose 5-phosphate, channeled through the pentose phosphate pathway and glycolysis.S. cerevisiae transformed with the XYL1 and XYL2 genes encoding xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis (referred to as PsXR and PsXDH, respectively) acquires the ability to ferment d-xylose to ethanol (2, 5, 6, 9, 10, 12, 22). Furthermore, overexpression of the XKS1 gene encoding xylulokinase (XK) from S. cerevisiae (ScXK) has been shown to aid d-xylose utilization (4, 7, 11, 16, 23), with xylitol still a major by-product. Kuyper et al. (14) also demonstrated the successful fermentation of d-xylose to ethanol using recombinant S. cerevisiae strains expressing fungal xylose isomerase. However, these approaches are insufficient for industrial bioprocesses, mainly due to the low rate of d-xylose fermentation.Xylitol excretion has been ascribed mainly to the difference in coenzyme specificities between PsXR (with NADPH) and PsXDH (with NAD⁺), which creates an intracellular redox imbalance (1). Therefore, modifying the coenzyme specificity of XR and/or XDH by protein engineering is an attractive approach for achieving efficient fermentation of ethanol from d-xylose using recombinant S. cerevisiae. Watanabe et al. (24) previously succeeded in generating several PsXDH mutants (e.g., quadruple ARSdR mutant) with a complete reversal of coenzyme specificity toward NADP⁺ by multiple site-directed mutagenesis on amino acids from the coenzyme-binding domain. The ARSdR mutant (D207A/I208R/F209S/N211R) has more that 4,500-fold-higher catalytic efficiency (k_cat/K_m) with NADP⁺ than the wild-type PsXDH. In addition, we recently found that several laboratory recombinant S. cerevisiae strains, in which the ARSdR mutant, along with PsXR and ScXK, is expressed through a strong constitutive promoter, increased ethanol production from d-xylose at the expense of xylitol excretion (17, 18), probably by maintaining the intracellular redox balance. However, commercialization of lignocellulosic hydrolysate fermentation requires industrial strains that are more robust than laboratory strains (5, 19, 21).A potential host for developing d-xylose-fermenting strains requires an active and efficient pentose phosphate pathway linking the d-xylose-to-d-xylulose pathway to glycolysis. In the case of S. cerevisiae, strains have different d-xylulose fermentation abilities (3, 25), indicating inherent differences in the capacities of these strains to ferment pentose sugars. Furthermore, anaerobic d-xylulose fermentation was investigated to identify genetic backgrounds potentially beneficial to anaerobic d-xylose fermentation in strains not exhibiting product formation related to the redox imbalance generated by the first two steps of the eukaryotic d-xylose metabolism (3), although the physiological purpose of the different d-xylulose fermentation abilities of S. cerevisiae is not yet clear. Therefore, we selected an efficient industrial d-xylulose-fermenting S. cerevisiae strain as a host for constructing a recombinant strain through chromosomal integration of the NADP⁺-dependent XDH gene and the XR and endogenous XK genes. Using this recombinant strain, we characterized the enzyme activity and ability to ferment both d-xylose and lignocellulosic hydrolysate.     

NADP+-Dependent internalization of recombinant CD38 in CHO cells

CD38 is a 46-kDa type II transmembrane glycoprotein that catalyses the synthesis of cyclic ADP-ribose (cADPR) from NAD+. cADPR is a second messenger known to regulate intracellular Ca2+-induced Ca2+-release (CICR). A recent study has revealed that CD38 in Namalwa B cells undergoes internalization upon exposure to external NAD+. In this study, recombinant rat CD38 was expressed in Chinese hamster ovary (CHO) cells and the possibility of the protein to undergo internalization upon exposure to a substrate analog NADP+ was examined. It was found that such treatment of CHO cells resulted in a decrease of ADP-ribosyl cyclase activity, as well as immunofluorescence of CD38 on the cell surface. The same treatment of CHO cells also resulted in intracellular clustering of CD38 molecules as revealed by confocal microscopic analysis. The internalized CD38 was purified using a streptavidin/biotin-based method and was found to exhibit both ADP-ribosyl cyclase and cADPR hydrolase activities. On immunoblot, the internalized CD38 appeared as a monomer of 46 kDa under reducing condition of SDS-PAGE. Our data demonstrate that NADP+ can efficiently induce internalization of CD38, a process that may be important in the production of cADPR intracellularly to regulate CICR.     

Sorbitol Synthesis in Transgenic Tobacco with Apple cDNA Encoding NADP-Dependent Sorbitol-6-Phosphate Dehydrogenase

The apple (Malus domestica) cDNA encoding NADP-dependent sorbitol-6-phosphatedehydrogenase (S6PDH) was stably integrated and expressed intransgenic tobacco (Nicotiana tabacum cv. SR1). Expression ofthe cDNA in either a sense or antisense orientation was accomplishedusing cauliflower mosaic virus regulatory sequences (CaMV35S).Sorbitol synthesis was confirmed by gas-chromatography-mass-spectroscopy(GC-MS). Sorbitol concentration in the leaves of the transgenicplants expressing the sense orientation varied from 186 to 446nmol (g fr wt)^-1. The concentration positively correlates withS6PDH activity in leaves. Neither sorbitol nor S6PDH activitywas detected in the extracts of nontransformed tobacco or transgenictobacco expressing the antisense orientation. These resultsprovide key genetic evidence that S6PDH expression is sufficientfor the synthesis of sorbitol in tobacco, implicating it asa key enzyme in the sorbitol biosynthetic pathway in apple andperhaps other members of the woody Rosaceae. ¹Present address: Laboratory of Pomology, Faculty of Agriculture,Kyoto University, Sakyo, Kyoto, 606-01 Japan     

MonoADP-Ribosylation of the NAD+-Dependent Alcohol Dehydrogenase from Entamoeba histolytica

The human parasite Entamoeba histolytica is an amitochondrial protozoan whose metabolism depends on glucose fermentation. Among the metabolic enzymes absolutely required for amoeba growth is the NAD⁺-dependent alcohol dehydrogenase (EhADH2). The polymeric form of EhADH2 was sedimented at 160,000g, and in this fraction we observed [³²P]-labeling of a 96-kDa protein under mono-ADP-ribosylation conditions with [³²P]NAD⁺. The [³²P]-labeled protein had the same molecular weight as the EhADH2 monomer. Because of the importance of monoADP-ribosylation in the regulation of many physiological processes, the aim of this study was to determine whether EhADH2 is ADP-ribosylated, and what would be the consequence of this modification on its alcohol and aldehyde dehydrogenase enzymatic activities. This study describes the ADP-ribosylation of EhADH2. This modification did not have an effect on the enzymatic activities, but it may regulate other functions of EhADH2.     

Alteration of Coenzyme Specificity of Lactate Dehydrogenase from Thermus thermophilus by Introducing the Loop Region of NADP(H)-Dependent Malate Dehydrogenase

Previously we found that replacement of seven amino acid residues in a loop region markedly shifted the coenzyme specificity of malate dehydrogenase from NAD(H) toward NADP(H). In the present study, we replaced the seven amino acid residues in the corresponding region of an NAD(H)-dependent lactate dehydrogenase with those of NADP(H)-dependent malate dehydrogenase, and examined the coenzyme specificity of the resulting mutant enzyme. Coenzyme specificity was significantly shifted by 399-fold toward NADPH when k _cat?K _m ^coenzyme was used as the measure of coenzyme specificity. The effect of the replacements on coenzyme specificity is discussed based on in silico simulation of the three-dimensional structure of the lactate dehydrogenase mutant.     

Alcohol Dehydrogenase of Apple

The alcohol dehydrogenase prepared from apple (Malus domesticaBorkh.) possesses both NADH and NADPH-linked activities, whenassayed with acetaldehyde as substrate. The pyridine nucleotidesbind to the same catalytic site on the enzyme. The alcohol dehydrogenasecan also catalyse the reduction of C₃–C₆ aldehydes witheither NADH or NADPH as cofactor.     

Comparative Studies of NAD+-Dependent Maltose Dehydrogenase and d-Glucose Dehydrogenase Produced by Two Strains of Alkalophilic Corynebacterium Species

Glycinin was dialyzed against low ionic strength buffer (μ = 0.01) and centrifuged in sucrose density gradient. Two major components with the sizes of 7S and 1 IS were obtained. When each component was separately recentrifuged, the intrinsic peak of each was predominantly given. This indicates that there were two molecular species in the glycinin, one being dissociable and the other undissociable at low ionic strength. The dissociable species reversibly associated to the size of 11S at high ionic strength. The conformation of each species was different, the dissociable species being more random and unstable than the undissociable species at low ionic strength. The dissociable species contained more ASIV and less ASIII than the undissociable species.     

Cellular Defense against Singlet Oxygen-induced Oxidative Damage by Cytosolic NADP + -dependent Isocitrate Dehydrogenase

Singlet oxygen ( 1 O 2 ) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. Recently, we have shown that NADP + -dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study, we investigated the role of cytosolic form of NADP + -dependent isocitrate dehydrogenase (IDPc) against singlet oxygen-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to singlet oxygen generated from photoactivated dye, the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against singlet oxygen, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against singlet oxygen-induced oxidative injury.     

Purification and Characterization of Sorbitol Dehydrogenase from Bovine Brain

Sorbitol dehydrogenase (EC 1.1.1.14) was isolated from bovine brain and purified 3,000-fold to apparent homogeneity, as judged by polyacrylamide gel electrophoresis. The purified enzyme had a specific activity of 36 units/mg of protein; a molecular weight of 39,000 for each of the four identical subunits and 155,000 for the intact enzyme were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel exclusion chromatography, respectively. The presence of one Zn2+ per subunit was confirmed by atom absorption spectroscopy; inactivation of the enzyme by metal-chelating agents points to the essential role that Zn2+ plays in the catalytically competent enzyme. The enzyme is also inactivated by thiol-blocking reagents; with respect to inactivation by sodium pyrophosphate, sorbitol dehydrogenase is different from closely related alcohol dehydrogenase.     

Nitrogen Source Regulates Glutamate Dehydrogenase NADP Synthesis in Neurospora crassa

Neurospora crassa glutamate dehydrogenase-NADP (EC 1.3.1.3) has a higher activity when mycelium is grown on ammonium or nitrate as nitrogen source than when grown on glutamate or glutamine. Quantitative immunoelectrophoresis established that, under all conditions, enzyme activity corresponded to enzyme concentration. Isotope incorporation studies demonstrated that the nitrogen source exerts its regulation at the level of de novo enzyme synthesis.     

Sorbitol in Tracheal Sap of Apple as Related to Temperature

The influence of dormant-season temperatures on levels of sorbitol was determined in an effort to provide further information on the possible role of sorbitol in dormancy of apple tress (Malus sylvestris Mill.). Two-year-old shoots were collected throughout the dormant season; sorbitol and sugars were determined in tracheal sap extracted under vacuum and in ground dried wood extracted in a soxhlet apparatus. Sorbitol and sugar trimethylsilyl derivatives were detected by gas chromatography. Levels of sorbitol in the sap generally increased during subfreezing temperatures and decreased during warm periods throughout the dormant season. Early peak increases in sap sorbitol appeared to coincide with the beginning of leaf senescence. Postharvest levels of reducing sugars, sorbitol, and particularly sucrose in the wood, increased as the temperature decreased during the dormant season, and the sugar levels decreased with warming temperatures in the spring. Our data indicate that sorbitol and sucrose are important reserves of storage carbohydrates in resting apple trees.     

The Effect of NADP+ on Electrophoretic Behavior of Glucose 6-Phosphate Dehydrogenase from Sweet Potato

The gene from Bacillus brevis TT02–8 encoding arginase was cloned into Escherichia coli, and its nucleotide sequence was identified. The nucleotide sequence contained an open reading frame that encoded a polypeptide of 298 amino acid residues with a predicted molecular weight of 31,891, which was consistent with that previously calculated for arginase purified from this bacterium. Comparison of the deduced amino acid sequence of the B. brevis TT02–8 arginase with that of the prokaryotic and eukaryotic arginases of Bacillus caldovelox, Bacillus subtilis, Agrobacterium Ti plasmid C58, Saccharomyces cerevisiae, Coccidioides immitis, Xenopus laevis, Rana catesbeiana, rat liver, and human liver, showed 33–66% of the sequences to be similar; there were several highly conserved regions. Arginase activity was detected in Escherichia coli cells transformed with an expression plasmid of the cloned arginase gene.     

一种人肝辅酶Ⅱ依赖性视黄醇脱氢酶剪接体cDNA的克隆及特征分析

在用RT-PCR法局部扩增人与小鼠肝脏635bp的NRDR DNA时,在人肝中发现了另一短序列PCR产物,克隆后测序显示其整个序列与NRDR cDNA编码区的前后序列完全一致。采用3’-Race和5’-Race方法,从人肝组织细胞中扩增得到两个全长cDNA,除1261bp的NRDR cDNA外,另一个为全长1003bp、编码区长为525bp的NRDRiso(GenBank登录号：AY071856)。数据库分析表明,NRDRiso编码区是由人NRDR8个外显子中的第1、2、3、7、8外显子选择性剪接而成。缺失的NRDR第4、5、6外显子共258bp,编码86个氨基酸。因此,与人NRDR的260个氨基酸残基相比,NRDRiso由174个氨基酸残基组成,分子量为18．6kDa,并且NRDRiso的组织表达与NRDR明显不同。     

ATP-Promoted Sorbitol Transport into Vacuoles Isolated from Apple Fruit

Sorbitol was transported actively into vacuoles isolated fromapple (Malus pumilla Mill, var domestica Schneid.) fruit flesh.The uptake was stimulated up to twofold by the addition of ATP,and the ATP dependent uptake showed a saturation curve as tothe substrate concentration. The optimum uptake of sorbitolwas pursued in the acidic range of pH 5 to 6. The K_m value forthe ATP dependent sorbitol uptake was about 5 mM. Sorbitol uptake was clearly inhibited by PCMB and uncouplers(CCCP and DCCD), and to a lesser extent by orthovanadate, butonly slightly by oligomycin. K⁺ stimulated sorbitol uptake.Sorbitol was converted to other sugars (glucose) only very slowlywhen transported across the tonoplast. This suggests that sorbitolis transported into vacuoles by a carrier mediated transportsystem coupled with H⁺- ATPase, localized on the tonoplast.Sucrose uptake into the vacuoles was also enhanced by ATP. (Received May 31, 1986; Accepted March 2, 1987)     

Purification and Characterization of an NAD+-Dependent XylB-Like Aryl Alcohol Dehydrogenase Identified in Acinetobacter baylyi ADP1

The gene xylB_ADP1 from Acinetobacter baylyi ADP1 (gene annotation number ACIAD1578), coding for a putative aryl alcohol dehydrogenase, was heterologously expressed in Escherichia coli BL21(DE3). The respective aryl alcohol dehydrogenase was purified by fast protein liquid chromatography to apparent electrophoretic homogeneity. The predicted molecular weight of 39,500 per subunit was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. According to the native M_w as determined by gel filtration, the enzyme forms dimers and therefore seems to be XylB related. The enzyme showed the highest activity at 40°C. For both the reduction and the oxidation reactions, the pH for optimum activity was 6.5. The enzyme was NADH dependent and able to reduce medium- to long-chain n-alkylaldehydes, methyl-branched aldehydes, and aromatic aldehydes, with benzaldehyde yielding the highest activity. The oxidation reaction with the corresponding alcohols showed only 2.2% of the reduction activity, with coniferyl alcohol yielding the highest activity. Maximum activities for the reduction and the oxidation reaction were 104.5 and 2.3 U mg⁻¹ of protein, respectively. The enzyme activity was affected by low concentrations of Ag⁺ and Hg²⁺ and high concentrations of Cu²⁺, Zn²⁺, and Fe²⁺. The gene xylB_ADP1 seems to be expressed constitutively and an involvement in coniferyl alcohol degradation is suggested. However, the enzyme is most probably not involved in the degradation of benzyl alcohol, anisalcohol, salicyl alcohol, vanillyl alcohol, cinnamyl alcohol, or aliphatic and isoprenoid alcohols.