Pyridine Nucleotide Cofactors Research Articles

Unbalance between Pyridine Nucleotide Cofactors in The SOD1 Deficient Yeast Saccharomyces cerevisiae Causes Hypersensitivity to Alcohols and Aldehydes.

Alcohol and aldehyde dehydrogenases are especially relevant enzymes involved in metabolic and detoxification reactions that occur in living cells. The comparison between the gene expression, protein content, and enzymatic activities of cytosolic alcohol and aldehyde dehydrogenases of the wild-type strain and the Δsod1 mutant lacking superoxide dismutase 1, which is hypersensitive to alcohols and aldehydes, shows that the activity of these enzymes is significantly higher in the Δsod1 mutant, but this is not a mere consequence of differences in the enzymatic protein content nor in the expression levels of genes. The analysis of the NAD(H) and NADP(H) content showed that the higher activity of alcohol and aldehyde dehydrogenases in the Δsod1 mutant could be a result of the increased availability of pyridine nucleotide cofactors. The higher level of NAD+ in the Δsod1 mutant is not related to the higher level of tryptophan; in turn, a higher generation of NADPH is associated with the upregulation of the pentose phosphate pathway. It is concluded that the increased sensitivity of the Δsod1 mutant to alcohols and aldehydes is not only a result of the disorder of redox homeostasis caused by the induction of oxidative stress but also a consequence of the unbalance between pyridine nucleotide cofactors.

Experimental Clarification of PRPS-1 Structural Essentials.

Phosphoribosyl pyrophosphate synthetase-1 (PRPS-1; EC 2.7.6.1.) catalyzes the binding of phosphate-group to ribose 5-phosphate, forming the 5-phosphoribosyl-1-pyrophosphate, which is necessary for the salvage pathways of purine and pyrimidine, pyridine nucleotide cofactors - NAD and NADP, the amino acids histidine and tryptophan biosynthesis. We aimed to investigate the impact of the different effectors on the activity of PRPS-1, to check the activity of the enzyme in vitro in a wide range of pHs and investigate some structural essentials of the enzyme, isolated from brain and liver. Molecular docking analyses were used to delineate the essentials of PRPS-1 structure, to find out the existence of enzyme effectors. Previously created by us kit was used for determination of the activity of PRPS-1 based on the formation of the inorganic phosphates (λ = 700 nm, Cary 60, Agilent, USA). Effectors impact on the activity of PRPS-1 was evaluated. In silico results of the effectors were later proven by in vitro experiments. For the first time biochemical essentials, including the existence of the multiple pockets, involvement of the amino acids into the processes of interactions with the effectors, evolutional of the sequence conservation, tissue depended Vmax differences were identified.

Linkage between Carbon Metabolism, Redox Status and Cellular Physiology in the Yeast Saccharomyces cerevisiae Devoid of SOD1 or SOD2 Gene.

Saccharomyces cerevisiae yeast cells may generate energy both by fermentation and aerobic respiration, which are dependent on the type and availability of carbon sources. Cells adapt to changes in nutrient availability, which entails the specific costs and benefits of different types of metabolism but also may cause alteration in redox homeostasis, both by changes in reactive oxygen species (ROS) and in cellular reductant molecules contents. In this study, yeast cells devoid of the SOD1 or SOD2 gene and fermentative or respiratory conditions were used to unravel the connection between the type of metabolism and redox status of cells and also how this affects selected parameters of cellular physiology. The performed analysis provides an argument that the source of ROS depends on the type of metabolism and non-mitochondrial sources are an important pool of ROS in yeast cells, especially under fermentative metabolism. There is a strict interconnection between carbon metabolism and redox status, which in turn has an influence on the physiological efficiency of the cells. Furthermore, pyridine nucleotide cofactors play an important role in these relationships.

Phosphomimetic T335D Mutation of Hydroxypyruvate Reductase 1 Modifies Cofactor Specificity and Impacts Arabidopsis Growth in Air.

Photorespiration is an essential process in oxygenic photosynthetic organisms triggered by the oxygenase activity of Rubisco. In peroxisomes, photorespiratory HYDROXYPYRUVATE REDUCTASE1 (HPR1) catalyzes the conversion of hydroxypyruvate to glycerate together with the oxidation of a pyridine nucleotide cofactor. HPR1 regulation remains poorly understood; however, HPR1 phosphorylation at T335 has been reported. By comparing the kinetic properties of phosphomimetic (T335D), nonphosphorylatable (T335A), and wild-type recombinant Arabidopsis (Arabidopsis thaliana) HPR1, it was found that HPR1-T335D exhibits reduced NADH-dependent hydroxypyruvate reductase activity while showing improved NADPH-dependent activity. Complementation of the Arabidopsis hpr1-1 mutant by either wild-type HPR1 or HPR1-T335A fully complemented the photorespiratory growth phenotype of hpr1-1 in ambient air, whereas HPR1-T335D-containing hpr1-1 plants remained smaller and had lower photosynthetic CO2 assimilation rates. Metabolite analyses indicated that these phenotypes were associated with subtle perturbations in the photorespiratory cycle of HPR1-T335D-complemented hpr1-1 rosettes compared to all other HPR1-containing lines. Therefore, T335 phosphorylation may play a role in the regulation of HPR1 activity in planta, although it was not required for growth under ambient air controlled conditions. Furthermore, improved NADP-dependent HPR1 activities in peroxisomes could not compensate for the reduced NADH-dependent HPR1 activity.

Production of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate using carbonyl reductase coupled with glucose dehydrogenase with high space-time yield.

tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is an important chiral intermediate for the synthesis of rosuvastatin. The biotechnological production of (3R,5S)-CDHH is catalyzed from tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH) by a carbonyl reductase, and this synthetic pathway is becoming a primary route for (3R,5S)-CDHH production due to its high enantioselectivity, mild reaction conditions, low cost, process safety, and environmental friendship. However, the requirement of the pyridine nucleotide cofactors, reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) limits its economic flexibility. In the present study, a recombinant Escherichia coli strain harboring carbonyl reductase R9M and glucose dehydrogenase (GDH) was constructed with high carbonyl reduction activity and cofactor regeneration efficiency. The recombinant E. coli cells were applied for the efficient production of (3R,5S)-CDHH with a substrate conversion of 98.8%, a yield of 95.6% and an enantiomeric excess (e.e.) of >99.0% under 350 g/L of (S)-CHOH after 12 hr reaction. A substrate fed-batch strategy was further employed to increase the substrate concentration to 400 g/L resulting in an enhanced product yield to 98.5% after 12 hr reaction in a 1 L bioreactor. Meanwhile, the space-time yield was 1,182.3 g L-1 day-1 , which was the highest value ever reported by a coupled system of carbonyl reductase and glucose dehydrogenase.

NADH oxidase from Lactobacillus reuteri: A versatile enzyme for oxidized cofactor regeneration

Pyridine nucleotide cofactors play important roles in biocatalytic processes that generate value-added chemicals for the pharmaceutical and food industries. Because of the high price of these pyridine cofactors, cofactor regeneration is highly desirable. However, recycling the oxidized form of cofactors, especially NADP+, remains a challenge. Here, we cloned and characterized an NADH oxidase from Lactobacillus reuteri (LreNox) which can oxidize both NADH and NADPH. Unlike many other Noxs, LreNox showed equal catalytic efficiency towards NADH and NADPH. To the best our knowledge, LreNox has the highest activity towards NADPH as a substrate compared to other wild type Noxs. Homology modeling and substrate docking studies provided insights into the dual substrate specificity of LreNox. Gly155, Ser179, and His184 in the LreNox substrate binding pocket, which are absent in other Noxs structures, are crucial for NADPH recognition, providing more space for interactions with the additional phosphate group present in NADPH. We also explored the utility of LreNox for NADP+ regeneration in l-sorbose production by coupling it with a sorbitol dehydrogenase. The turn over number (TTN) improved ~53-fold after using LreNox as the NADP+ recycling enzyme. This study demonstrates that LreNox could potentially be used for the regeneration of NAD(P)+ in commercial applications.

Mouse Nudt13 is a Mitochondrial Nudix Hydrolase with NAD(P)H Pyrophosphohydrolase Activity

The mammalian NUDT13 protein possesses a sequence motif characteristic of the NADH pyrophosphohydrolase subfamily of Nudix hydrolases. Due to the persistent insolubility of the recombinant product expressed in Escherichia coli, active mouse Nudt13 was expressed in insect cells from a baculovirus vector as a histidine-tagged recombinant protein. In vitro, it efficiently hydrolysed NADH to NMNH and AMP and NADPH to NMNH and 2′,5′-ADP and had a marked preference for the reduced pyridine nucleotides. Much lower activity was obtained with other nucleotide substrates tested. Km and kcat values for NADH were 0.34 mM and 7 s−1 respectively. Expression of Nudt13 as an N-terminal fusion to green fluorescent protein revealed that it was targeted exclusively to mitochondria by the N-terminal targeting peptide, suggesting that Nudt13 may act to regulate the concentration of mitochondrial reduced pyridine nucleotide cofactors and the NAD(P)+/NAD(P)H ratio in this organelle and elsewhere. Future studies of the enzymology of pyridine nucleotide metabolism in relation to energy homeostasis, redox control, free radical production and cellular integrity should consider the possible regulatory role of Nudt13.

Heteromeric complexes of aldo-keto reductase auxiliary KVβ subunits (AKR6A) regulate sarcolemmal localization of KV1.5 in coronary arterial myocytes

Redox-sensitive potassium channels consisting of the voltage-gated K+ (KV) channel pore subunit KV1.5 regulate resting membrane potential and thereby contractility of vascular smooth muscle cells. Members of the KV1 family associate with cytosolic auxiliary β subunits, which are members of the aldo-keto reductase (AKR) superfamily (AKR6A subfamily). The Kvβ subunits have been proposed to regulate Kv1 gating via pyridine nucleotide cofactor binding. However, the molecular identity of KVβ subunits that associate with native KV1.5 channels in the vasculature is unknown. Here, we examined mRNA and protein expression of KVβ subunits and tested whether KVβ isoforms interact with KV1.5 channels in murine coronary arteries. We detected KVβ1 (AKR6A3), KVβ2 (AKR6A5) and KVβ3 (AKR6A9) transcripts and KVβ1 and KVβ2 protein in left anterior descending coronary arteries by real time quantitative PCR and Western blot, respectively. In situ proximity ligation assays indicated abundant protein-protein interactions between KV1.5/KVβ1, KV1.5/KVβ2 and KVβ1/β2 in coronary arterial myocytes. Confocal microscopy and membrane fractionation analyses suggest that arterial myocytes from KVβ2-null mice have reduced abundance of sarcolemmal KV1.5. Together, data suggest that in coronary arterial myocytes, KV1.5 channels predominantly associate with KVβ1 and KVβ2 proteins and that KVβ2 performs a chaperone function for KV1.5 channels in arterial myocytes, thereby facilitating Kv1α trafficking and membrane localization.

Selective Detection of NADPH among Four Pyridine‐Nucleotide Cofactors by a Fluorescent Probe Based on Aggregation‐Induced Emission

A fluorescent sensor based on guanidinium-tethered tetraphenylethene (TPE) has been investigated toward the differentiation of pyridine nucleotide cofactors (NAD(+) , NADH, NADP(+) , and NADPH). TPE selectively recognizes NADPH possessing the higher tetra-anionic net-charge, resulting in the steep "turn-on" fluorescence increase. The comparative aggregation behaviors and fluorescence response studies of TPE on the four cofactors reveal that the critical aggregate concentration of TPE against NADPH correlates directly with the concentration threshold for the fluorescence response. These results establish that TPE can selectively differentiate NADPH over the other three cofactors by the steep aggregation-induced fluorescence response accompanied by the high signal-to-background contrast.

A novel whole-cell biocatalyst with NAD+ regeneration for production of chiral chemicals.

BackgroundThe high costs of pyridine nucleotide cofactors have limited the applications of NAD(P)-dependent oxidoreductases on an industrial scale. Although NAD(P)H regeneration systems have been widely studied, NAD(P)+ regeneration, which is required in reactions where the oxidized form of the cofactor is used, has been less well explored, particularly in whole-cell biocatalytic processes.Methodology/Principal FindingsSimultaneous overexpression of an NAD+ dependent enzyme and an NAD+ regenerating enzyme (H2O producing NADH oxidase from Lactobacillus brevis) in a whole-cell biocatalyst was studied for application in the NAD+-dependent oxidation system. The whole-cell biocatalyst with (2R,3R)-2,3-butanediol dehydrogenase as the catalyzing enzyme was used to produce (3R)-acetoin, (3S)-acetoin and (2S,3S)-2,3-butanediol.Conclusions/SignificanceA recombinant strain, in which an NAD+ regeneration enzyme was coexpressed, displayed significantly higher biocatalytic efficiency in terms of the production of chiral acetoin and (2S,3S)-2,3-butanediol. The application of this coexpression system to the production of other chiral chemicals could be extended by using different NAD(P)-dependent dehydrogenases that require NAD(P)+ for catalysis.

Expansion of the aspartate β-semialdehyde dehydrogenase family: the first structure of a fungal ortholog

The enzyme aspartate semialdehyde dehydrogenase (ASADH) catalyzes a critical transformation that produces the first branch-point intermediate in an essential microbial amino-acid biosynthetic pathway. The first structure of an ASADH isolated from a fungal species (Candida albicans) has been determined as a complex with its pyridine nucleotide cofactor. This enzyme is a functional dimer, with a similar overall fold and domain organization to the structurally characterized bacterial ASADHs. However, there are differences in the secondary-structural elements and in cofactor binding that are likely to cause the lower catalytic efficiency of this fungal enzyme. Alterations in the dimer interface, through deletion of a helical subdomain and replacement of amino acids that participate in a hydrogen-bonding network, interrupt the intersubunit-communication channels required to support an alternating-site catalytic mechanism. The detailed functional information derived from this new structure will allow an assessment of ASADH as a possible target for antifungal drug development.

A novel NADP +-dependent dehydrogenase activity for 7α/β- and 11β-hydroxysteroids in human liver nuclei: A third 11β-hydroxysteroid dehydrogenase

Human tissue from uninvolved liver of cancer patients was fractionated using differential centrifugation and characterized for 11βHSD enzyme activity against corticosterone, dehydrocorticosterone, 7α- and 7β-hydroxy-dehydroepiandrosterone, and 7-oxo-dehydroepiandrosterone. An enzyme activity was observed in nuclear protein fractions that utilized either NADP + or NAD +, but not NADPH and NADH, as pyridine nucleotide cofactor with K m values of 12 ± 2 and 390 ± 2 μM, compared to the K m for microsomal 11βHSD1 of 43 ± 8 and 264 ± 24 μM, respectively. The K m for corticosterone in the NADP +-dependent nuclear oxidation reaction was 102 ± 16 nM, compared to 4.3 ± 0.8 μM for 11βHSD1. The K cat values for nuclear activity with NADP + was 1687 nmol/min/mg/μmol, compared to 755 nmol/min/mg/μmol for microsomal 11βHSD1 activity. Inhibitors of 11βHSD1 decreased both nuclear and microsomal enzyme activities, suggesting that the nuclear activity may be due to an enzyme similar to 11βHSD Type 1 and 2.

Dependence of amino acid side chain 13C shifts on dihedral angle: application to conformational analysis.

Chemical shift data from the BiomagResDataBank and conformational data derived from the protein data bank have been correlated in order to explore the conformational dependence of side chain (13)C resonance shifts. Consistent with predictions based on steric compression, upfield shifts for Cgamma resonances of Thr, Val, Ile, Leu, Met, Arg, Lys, Glu, and Gln residues correlate with both the number of heavy atom (nonproton) gamma-substituents and with gauche conformational orientations of gamma-substituents. The (13)C shift/conformation correlations are most apparent for Cgamma carbons but also can be observed at positions further from the backbone. Intraresidue steric conflict leads to a correlation between upfield-shifted side chain (13)C resonances and statistically lower probabilities in surveys of protein side chain conformation. Illustrative applications to the DNA pol lambda lyase domain and to dihydrofolate reductase are discussed. In the latter case, (13)C shift analysis indicates that the conformation of the remote residue V119 on the betaF-betaG loop is correlated with the redox state of the bound pyridine nucleotide cofactor, providing one basis for discrimination between substrate and product. It is anticipated that (13)C shift data for protein sidechains can provide a useful basis for the analysis of conformational changes even in large, deuterated proteins. Additionally, the large dependence of the leucine methyl shift difference, deltaCdelta1-deltaCdelta2, on both chi1 and chi2 is sufficient to allow this parameter to be used as a restraint in structure calculations if stereospecific assignment data are available.

In Vitro Characterization of AtsB, a Radical SAM Formylglycine-Generating Enzyme That Contains Three [4Fe-4S] Clusters

Sulfatases catalyze the cleavage of a variety of cellular sulfate esters via a novel mechanism that requires the action of a protein-derived formylglycine cofactor. Formation of the cofactor is catalyzed by an accessory protein and involves the two-electron oxidation of a specific cysteinyl or seryl residue on the relevant sulfatase. Although some sulfatases undergo maturation via mechanisms in which oxygen serves as an electron acceptor, AtsB, the maturase from Klebsiella pneumoniae, catalyzes the oxidation of Ser72 on AtsA, its cognate sulfatase, via an oxygen-independent mechanism. Moreover, it does not make use of pyridine or flavin nucleotide cofactors as direct electron acceptors. In fact, AtsB has been shown to be a member of the radical S-adenosylmethionine superfamily of proteins, suggesting that it catalyzes this oxidation via an intermediate 5'-deoxyadenosyl 5'-radical that is generated by a reductive cleavage of S-adenosyl- l-methionine. In contrast to AtsA, very little in vitro characterization of AtsB has been conducted. Herein we show that coexpression of the K. pneumoniae atsB gene with a plasmid that encodes genes that are known to be involved in iron-sulfur cluster biosynthesis yields soluble protein that can be characterized in vitro. The as-isolated protein contained 8.7 +/- 0.4 irons and 12.2 +/- 2.6 sulfides per polypeptide, which existed almost entirely in the [4Fe-4S] (2+) configuration, as determined by Mossbauer spectroscopy, suggesting that it contained at least two of these clusters per polypeptide. Reconstitution of the as-isolated protein with additional iron and sulfide indicated the presence of 12.3 +/- 0.2 irons and 9.9 +/- 0.4 sulfides per polypeptide. Subsequent characterization of the reconstituted protein by Mossbauer spectroscopy indicated the presence of only [4Fe-4S] clusters, suggesting that reconstituted AtsB contains three per polypeptide. Consistent with this stoichiometry, an as-isolated AtsB triple variant containing Cys --> Ala substitutions at each of the cysteines in its CX 3CX 2C radical SAM motif contained 7.3 +/- 0.1 irons and 7.2 +/- 0.2 sulfides per polypeptide while the reconstituted triple variant contained 7.7 +/- 0.1 irons and 8.4 +/- 0.4 sulfides per polypeptide, indicating that it was unable to incorporate an additional cluster. UV-visible and Mossbauer spectra of both samples indicated the presence of only [4Fe-4S] clusters. AtsB was capable of catalyzing multiple turnovers and exhibited a V max/[E T] of approximately 0.36 min (-1) for an 18-amino acid peptide substrate using dithionite to supply the requisite electron and a value of approximately 0.039 min (-1) for the same substrate using the physiologically relevant flavodoxin reducing system. Simultaneous quantification of formylglycine and 5'-deoxyadenosine as a function of time indicates an approximate 1:1 stoichiometry. Use of a peptide substrate in which the target serine is changed to cysteine also gives rise to turnover, supporting approximately 4-fold the activity of that observed with the natural substrate.

The B′ Helix Determines Cytochrome P450nor Specificity for the Electron Donors NADH and NADPH

Nitric oxide reductase (Nor) cytochrome P450nor (P450nor) is unique because it is catalytically self-sufficient, receiving electrons directly from NADH or NADPH. However, little is known about the direct binding of NADH to cytochrome. Here, we report that oxidized pyridine nucleotides (NAD(+) and NADP(+)) and an analogue induce a spectral perturbation in bound heme when mixed with P450nor. The P450nor isoforms are classified according to electron donor specificity for NADH or NADPH. One type (Fnor, a P450nor of Fusarium oxysporum) utilizes only NADH. We found that NAD(+) induced a type I spectral change in Fnor, whereas NADP(+) induced a reverse type I spectral change, although the K(d) values for both were comparable. In contrast, NADP(+) as well as NAD(+) caused a type I spectral change in Tnor, a P450nor isozyme from Trichosporon cutaneum that utilizes both NADH and NADPH as electron donors. The B' helix region of Tnor ((73)SAGGKAAA(80)) contains some Ala and Gly residues, whereas the sequence is replaced at a few sites with more bulky amino acid residues in Fnor ((73)SASGKQAA(80)). A single mutation (S75G) significantly improved the NADPH- dependent Nor activity of Fnor, and the overall activity was accelerated via the NADPH-enhanced reduction step. These results showed that pyridine nucleotide cofactors can bind P450nor and that only a few residues in the B' helix region determine cofactor specificity. We further showed that a poor electron donor (NADPH) could also bind Fnor, but an appropriate configuration for electron transfer is blocked by steric hindrance mainly by Ser(75) against the 2'-phosphate moiety. The present results along with previous observations together revealed a novel motif for cofactor binding.

Synthesis of pyridine nucleotide analogs consisting of nicotinoylamino acids by means of transglycosidation reactions catalyzed by mammalian pyridine nucleotide transglycosidases.

Besides isonicotinic acid analogs of pyridine nucleotides, 24 novel pyridine nucleotide cofactors that have an amino acid residue at the carbonyl carbon of the nicotinamide moiety have been prepared by means of transglycosidation reactions catalyzed by rabbit spleen and guinea pig spleen pyridine nucleotide transglycosidases. Their chemical properties were characterized by means of proton NMR, Fab-mass, and UV spectral measurement and phosphodiesterase digestion. Except for the isonicotinic acid ones, these nicotinoylamino acid analogs were shown to function as substrates for both the hydrolysis and the transglycosidation reactions catalyzed by the mammalian NAD glycohydrolases, though their substrate activities were lower than those with the original pyridine nucleotides (NMN, NAD, and NADP). They were inactive in regard to yeast alcohol dehydrogenase- and Thermoanaerobium brockii alcohol dehydrogenase (NADP dependent)-oxidation.

Characterization of a mechanism-based inhibitor of NAD(P)H:quinone oxidoreductase 1 by biochemical, X-ray crystallographic, and mass spectrometric approaches.

We report the characterization of 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936) as a mechanism-based inhibitor of NQO1. Inactivation of NQO1 by ES936 was time- and concentration-dependent and required the presence of a pyridine nucleotide cofactor consistent with a need for metabolic activation. That ES936 was an efficient inhibitor was demonstrated in these studies by the low partition ratio (1.40 +/- 0.03). The orientation of ES936 in the active site of NQO1 was examined by X-ray crystallography and found to be opposite to that observed for other indolequinones acting as substrates. ES936 was oriented in such a manner that, after enzymatic reduction and loss of a nitrophenol leaving group, a reactive iminium species was located in close proximity to nucleophilic His 162 and Tyr 127 and Tyr 129 residues in the active site. To determine if ES936 was covalently modifying NQO1, ES936-treated protein was analyzed by electrospray ionization liquid chromatography/mass spectrometry (ESI-LC/MS). The control NQO1 protein had a mass of 30864 +/- 6 Da (n = 20, theoretical, 30868.6 Da) which increased by 217 Da after ES936 treatment (31081 +/- 7 Da, n = 20) in the presence of NADH. The shift in mass was consistent with adduction of NQO1 by the reactive iminium derived from ES936 (M + 218 Da). Chymotryptic digestion of the protein followed by LC/MS analysis located a tetrapeptide spanning amino acids 126-129 which was adducted with the reactive iminium species derived from ES936. LC/MS/MS analysis of the peptide fragment confirmed adduction of either Tyr 127 or Tyr 129 residues. This work demonstrates that ES936 is a potent mechanism-based inhibitor of NQO1 and may be a useful tool in defining the role of NQO1 in cellular systems and in vivo.

Accumulation of α-oxoaldehydes during oxidative stress: a role in cytotoxicity

Glyoxal, methylglyoxal (MG), and 3-deoxyglucosone (3-DG) are physiological α-oxoaldehydes formed by lipid peroxidation, glycation, and degradation of glycolytic intermediates. They are enzymatically detoxified in cells by the cytosolic glutathione-dependent glyoxalase system (glyoxal and MG only) and by NADPH-dependent reductase and NAD(P) +-dependent dehydrogenase. In this study, the changes in the cellular and extracellular concentrations of these α-oxoaldehydes were investigated in murine P388D 1 macrophages during necrotic cell death induced by median toxic concentrations of hydrogen peroxide and 1-chloro-2,4-dinitrobenzene (CDNB). α-Oxoaldehyde concentrations were determined by derivatization with 1,2-diamino-4,5-dimethoxybenzene. There were relatively small increases in cellular and extracellular glyoxal concentration, except that extracellular glyoxal was decreased with hydrogen peroxide. The cytosolic concentration of 3-DG and the cytosolic and extracellular concentrations of MG, however, increased markedly. Aminoguanidine inhibited α-oxoaldehyde accumulation and prevented cytotoxicity induced by hydrogen peroxide and CDNB. The accumulation of glyoxal and MG in toxicant-treated cells was a likely consequence of decreased in situ activity of glyoxalase I. The effect was marked for MG but not for glyoxal, suggestive of a greater metabolic flux of MG formation than of glyoxal. The accumulation of 3-DG in toxicant-treated cells was probably due to the decreased availability of pyridine nucleotide cofactors for the detoxification of 3-DG. Impairment of α-oxoaldehyde detoxification is cytotoxic, and this may contribute to toxicity associated with GSH oxidation and S conjugation in oxidative stress and chemical toxicity, and to chronic pathogenesis associated with diabetes mellitus where there is oxidative stress and the formation of glyoxal, MG, and 3-DG is increased.

Purification to Homogeneity and Reconstitution of the Individual Components of the Epoxide Carboxylase Multiprotein Enzyme Complex from Xanthobacter Strain Py2

Epoxide metabolism in the aerobic bacterium Xanthobacter strain Py2 proceeds by an NADPH- and NAD+-dependent carboxylation reaction that forms beta-keto acids as products. Epoxide carboxylase, the enzyme catalyzing this reaction, was resolved from the soluble fraction of cell-free extracts into four protein components that are obligately required for functional reconstitution of epoxide carboxylase activity. One of these components, component II, has previously been purified and characterized as an NADPH:disulfide oxidoreductase. In the present study, the three additional epoxide carboxylase components have been purified to homogeneity and characterized. These component proteins are as follows: component I, a homohexameric protein consisting of 41.7-kDa subunits; component III, a dimeric protein consisting of 26.0- and 26.2-kDa polypeptides; and component IV, a dimeric protein consisting of a single 25.4-kDa polypeptide. Component I contained 5 mol of tightly bound zinc per mol of protein. Component I was specifically inactivated by methylepoxypropane, a time-dependent irreversible inactivator of epoxide carboxylase activity, suggesting that this component plays an integral role in epoxide binding and activation. No metals or organic cofactors were detected for components III and IV. The molecular weights, N-terminal sequences, and amino acid compositions of the purified epoxide carboxylase components were determined and found to correlate with open reading frames within and adjacent to a cloned fragment of DNA that complements Xanthobacter Py2 mutants defective in epoxide degradation. Using the purified epoxide carboxylase system, epoxide carboxylation was found to be stoichiometrically coupled to the transhydrogenation of pyridine nucleotide cofactors according to the following equation: epoxypropane + CO2 + NADPH + NAD+ --> acetoacetate + H+ + NADP+ + NADH.

Metabolic implications of stress-induced proline accumulation in plants

In many plants, free proline accumulates in response to the imposition of a wide range of biotic and abiotic stresses. Controversy has surrounded the extent to which this shift in nitrogen metabolism benefits plants under adverse environmental conditions. Most attempts to account for the phenomenon have focused on the ability of proline to mediate osmotic adjustment, stabilise subcellular structures and scavenge free radicals. However, often the cytoplasmic pool of free proline even after the imposition of stress is insufficient size to account for pronounced biophysical effects. Alternatively, selective preservation of this stress-induced response may relate to endpoints other than simply augmenting the cellular pool of free proline. Proline accumulation may reduce stress-induced cellular acidification or prime oxidative respiration to provide energy needed for recovery. High levels of proline synthesis during stress may maintain NAD(P)+/NAD(P)H ratios at values compatible with metabolism under normal conditions. Consideration of the cofactor preference of plant Δ1-pyrroline-5-carboxylate (P5C) reductase as well as the in vivo concentrations of the two pyridine nucleotide cofactors and their respective redox ratios suggests that even a small increase in proline biosynthesis might have a large impact on the level of reduction of the cellular NADP pool. The increased NADP+/NADPH ratio mediated by proline biosynthesis is likely to enhance activity of the oxidative pentose phosphate pathway. This would provide precursors to support the demand for increased secondary metabolite production during stress as well as nucleotide synthesis accompanying the accelerated rate of cell division upon relief from stress, when oxidation of proline is likely to provide an important energy source for ADP phosphorylation. Thus, the extreme sensitivity of the metabolic processes of proline synthesis and degradation themselves may be of benefit by regulating metabolic processes adversely affected by stress. This viewpoint is supported by consideration of other physiological phenomena not directly related to stress responses, but in which proline metabolism may also play a regulatory role. A mechanism is proposed whereby the interconversions of proline and P5C in different cell types and the associated transfer of redox potential between tissues may constitute a form of metabolic signalling within higher plants. Stress-related alterations in proline metabolism may impinge on systems of redox control of plant gene expression.