Salvage pathway for NAD biosynthesis in Brevibacterium ammoniagenes: regulatory properties of triphosphate-dependent nicotinate phosphoribosyltransferase

As the rate-limiting enzyme, catalyzing the first reaction in NAD salvage synthesis, nicotinate phosphoribosyltransferase (NAPRTase, EC 2.4.2.11) is of important interest for studies of intracellular pyridine nucleotide pool regulation. We have purified NAPRTase 520-fold from Brevibacterium ammoniagenes ATCC 6872 without using an over-expression system by applying acid treatment, salt fractionation, Ca-phosphate gel treatment, anion exchange column chromatography and size-exclusion gel filtration. Unlike this enzyme from other sources, B. ammoniagenes NAPRTase was found to be controlled by the feedback inhibition by the end product NAD with K(i)=0.7+/-0.1 mM. The reaction products, pyrophosphate and nicotinate mononucleotide, also decreased the enzyme activity, as did other intermediates of NAD synthesis, such as AMP, ADP and a NAD direct precursor, nicotinate adenine dinucleotide or deamido NAD. The enzyme was observed to require a nucleoside triphosphate for its activity and showed the maximum affinity for ATP. The specificity, however, turned out to be poor, and ATP could be substituted by other nucleoside triphosphates as well as by sodium triphosphate. The kinetic characteristics of the enzyme are reported. For the first time, our data have experimentally revealed such complicated stimulatory and inhibitory effects by the intermediates of NAD biosynthesis on one of its salvage enzymes, NAPRTase. On the basis of these data, the key role of NAPRTase is discussed in light of the regulation of NAD metabolism in B. ammoniagenes.     

Crystal structure of a nicotinate phosphoribosyltransferase from Thermoplasma acidophilum

We have determined the crystal structure of nicotinate phosphoribosyltransferase from Themoplasma acidophilum (TaNAPRTase). The TaNAPRTase has three domains, an N-terminal domain, a central functional domain, and a unique C-terminal domain. The crystal structure revealed that the functional domain has a type II phosphoribosyltransferase fold that may be a common architecture for both nicotinic acid and quinolinic acid (QA) phosphoribosyltransferases (PRTase) despite low sequence similarity between them. Unlike QAPRTase, TaNAPRTase has a unique extra C-terminal domain containing a zinc knuckle-like motif containing 4 cysteines. The TaNAPRTase forms a trimer of dimers in the crystal. The active site pocket is formed at dimer interfaces. The complex structures with phosphoribosylpyrophosphate (PRPP) and nicotinate mononucleotide (NAMN) showed, surprisingly, that functional residues lining on the active site of TaNAPRTase are quite different from those of QAPRTase, although their substrates are quite similar to each other. The phosphate moiety of PRPP and NAMN is anchored to the phosphate-binding loops formed by backbone amides, as found in many alpha/beta barrel enzymes. The pyrophosphate moiety of PRPP is located at the entrance of the active site pocket, whereas the nicotinate moiety of NAMN is located deep inside. Interestingly, the nicotinate moiety of NAMN is intercalated between highly conserved aromatic residues Tyr(21) and Phe(138). Careful structural analyses combined with other NAPRTase sequence subfamilies reveal that TaNAPRTase represents a unique sequence subfamily of NAPRTase. The structures of TaNAPRTase also provide valuable insight for other sequence subfamilies such as pre-B cell colony-enhancing factor, known to have nicotinamide phosphoribosyltransferase activity.     

Stimulation of the Na+,K(+)-ATPase activity of K562 human erythroleukemia cells by triiodothyronine.

The effect of triiodothyronine (T3) on Na+,K(+)-ATPase activity of K562 human erythroleukemic cell was studied to understand why the erythrocyte sodium pump activity is decreased in hyperthyroidism. Na+,K(+)-ATPase activity of K562 cell lysates was assayed by measuring the release of inorganic phosphate (Pi) from ATP. Na+,K(+)-ATPase activity of K562 cell grown in the presence of T3 for 48 hours was significantly higher than that of control (0.98 +/- 0.05 mumol Pi h-1 mg protein-1 vs 0.82 +/- 0.10 mumol Pi h-1 mg protein-1, p < 0.05). The Na+,K(+)-ATPase activity could be stimulated in a time- and concentration-dependent manner; maximum stimulatory effect of T3 was seen at a concentration of 10(-7) mol/L. When an inducer [cytosine-beta-D-arabino-furanoside (ARA-C)] was added to the culture medium, the K562 cells showed signs of differentiation and synthesised haemoglobin. At the same time, the Na+,K(+)-ATPase activity remained high. We conclude that T3 stimulates Na+,K(+)-ATPase activity of K562 cells and in the presence of T3 during differentiation, the enzyme activity remains high.     

Nitric oxide -- superoxide cooperation in the regulation of renal Na(+),K(+)-ATPase

The aim of this study was to investigate whether endogenous superoxide anion is involved in the regulation of renal Na(+),K(+)-ATPase and ouabain-sensitive H(+),K(+)-ATPase activities. The study was performed in male Wistar rats. Compounds modulating superoxide anion concentration were infused under general anaesthesia into the abdominal aorta proximally to the renal arteries. The activity of ATPases was assayed in isolated microsomal fraction. We found that infusion of a superoxide anion-generating mixture, xanthine oxidase (1 mU/min per kg) + hypoxanthine (0.2 mumol/min per kg), increased the medullary Na(+),K(+)-ATPase activity by 49.5% but had no effect on cortical Na(+),K(+)-ATPase and either cortical or medullary ouabain-sensitive H(+),K(+)-ATPase. This effect was reproduced by elevating endogenous superoxide anion with a superoxide dismutase inhibitor, diethylthiocarbamate. In contrast, a superoxide dismutase mimetic, TEMPOL, decreased the medullary Na(+),K(+)-ATPase activity. The inhibitory effect of TEMPOL was abolished by inhibitors of nitric oxide synthase (L-NAME), soluble guanylate cyclase (ODQ) and protein kinase G (KT5823). The stimulatory effect of diethylthiocarbamate was not observed in animals pretreated with a synthetic cGMP analogue, 8-bromo-cGMP. An inhibitor of NAD(P)H oxidase, apocynin (1 mumol/min per kg), decreased the Na(+),K(+)-ATPase activity in the renal medulla and its effect was prevented by L-NAME, ODQ or KT5823. In contrast, a xanthine oxidase inhibitor, oxypurinol, administered at the same dose was without effect. These data suggest that NAD(P)H oxidase-derived superoxide anion increases Na(+),K(+)-ATPase activity in the renal medulla by reducing the availability of NO. Excessive intrarenal generation of superoxide anion may upregulate medullary Na(+),K(+)-ATPase leading to sodium retention and blood pressure elevation.     

Structure of human nicotinamide/nicotinic acid mononucleotide adenylyltransferase. Basis for the dual substrate specificity and activation of the oncolytic agent tiazofurin.

Nicotinamide/nicotinate mononucleotide (NMN/ NaMN)adenylyltransferase (NMNAT) is an indispensable enzyme in the biosynthesis of NAD(+) and NADP(+). Human NMNAT displays unique dual substrate specificity toward both NMN and NaMN, thus flexible in participating in both de novo and salvage pathways of NAD synthesis. Human NMNAT also catalyzes the rate-limiting step of the metabolic conversion of the anticancer agent tiazofurin to its active form tiazofurin adenine dinucleotide (TAD). The tiazofurin resistance is mainly associated with the low NMNAT activity in the cell. We have solved the crystal structures of human NMNAT in complex with NAD, deamido-NAD, and a non-hydrolyzable TAD analogue beta-CH(2)-TAD. These complex structures delineate the broad substrate specificity of the enzyme toward both NMN and NaMN and reveal the structural mechanism for adenylation of tiazofurin nucleotide. The crystal structure of human NMNAT also shows that it forms a barrel-like hexamer with the predicted nuclear localization signal sequence located on the outside surface of the barrel, supporting its functional role of interacting with the nuclear transporting proteins. The results from the analytical ultracentrifugation studies are consistent with the formation of a hexamer in solution under certain conditions.     

NADH/NAD redox state of cytoplasmic glycolytic compartments in vascular smooth muscle

The cytoplasmic NADH/NAD redox potential affects energy metabolism and contractile reactivity of vascular smooth muscle. NADH/NAD redox state in the cytosol is predominately determined by glycolysis, which in smooth muscle is separated into two functionally independent cytoplasmic compartments, one of which fuels the activity of Na(+)-K(+)-ATPase. We examined the effect of varying the glycolytic compartments on cystosolic NADH/NAD redox state. Inhibition of Na(+)-K(+)-ATPase by 10 microM ouabain resulted in decreased glycolysis and lactate production. Despite this, intracellular concentrations of the glycolytic metabolite redox couples of lactate/pyruvate and glycerol-3-phosphate/dihydroxyacetone phosphate (thus NADH/NAD) and the cytoplasmic redox state were unchanged. The constant concentration of the metabolite redox couples and redox potential was attributed to 1) decreased efflux of lactate and pyruvate due to decreased activity of monocarboxylate B-H(+) transporter secondary to decreased availability of H(+) for cotransport and 2) increased uptake of lactate (and perhaps pyruvate) from the extracellular space, probably mediated by the monocarboxylate-H(+) transporter, which was specifically linked to reduced activity of Na(+)-K(+)-ATPase. We concluded that redox potentials of the two glycolytic compartments of the cytosol maintain equilibrium and that the cytoplasmic NADH/NAD redox potential remains constant in the steady state despite varying glycolytic flux in the cytosolic compartment for Na(+)-K(+)-ATPase.     

Identification of the roles of conserved charged residues in the extracellular domain of an epithelial sodium channel (ENaC) subunit by alanine mutagenesis

Epithelial sodium channels (ENaC) are composed of three homologous subunits whose extracellular domains (ECD) form a funnel that directs ions from the lumen into the pore of ENaC. To examine the roles of conserved charged residues (Asp, Glu, Arg, and Lys) on ECD, we mutated 16 residues in human α-ENaC to alanine. The modified cRNAs were expressed in Xenopus laevis oocytes together with wild-type β- and γ-ENaC. The effect of each mutation was examined on three parameters: amiloride-sensitive Na(+) conductance (assayed by the two-electrode voltage-clamp method), Na(+)-dependent self-inhibition of ENaC, and oocyte cell surface expression of ENaC (quantitated by confocal microscopy of yellow fluorescent protein linked to γ-ENaC). Mutation of 13 of 16 residues reduced the ENaC Na(+) conductance (to 40-80% of WT). Mutation of only six residues showed a significant effect on the Na(+) self-inhibition time constant (τ). All 16 mutants showed a strong correlation between ENaC activity and oocyte surface expression (r = 0.62). Exclusion of four mutants showing the greatest effect on self-inhibition kinetics (Glu250 and Arg350 with τ = ~30% of WT, and Asp393 and Glu530 with τ = ~170% of WT) increased the correlation to r = 0.87. In the ASIC1 homotrimeric model, the homologs of α-ENaC Asp400 and Asp446 are exposed on the protein surface far from the other two chains. The mutations of these two residues showed the strongest effect on cell surface expression but had no effect on self-inhibition. Control mutations to a homologous charged residue (e.g., Asp to Glu) did not significantly affect ENaC activity. Changes in the two parameters, Na(+) self-inhibition and oocyte surface expression level, accounted for the magnitude of reduction in ENaC activity as a result of the mutation to Ala. These results establish that while some conserved charged residues are part of the structure responsible for Na(+) self-inhibition, most are essential for transport to the oocyte cell surface.     

Arabidopsis thaliana nicotinate/nicotinamide mononucleotide adenyltransferase (AtNMNAT) is required for pollen tube growth

While mammals and fungi possess nicotinate/nicotinamide mononucleotide adenyltransferase (NMNAT) isoforms, Arabidopsis thaliana only contains a single NMNAT gene, AtNMNAT (At5g55810). We analyzed the enzymatic activity of the AtNMNAT-encoded protein to determine the role of AtNMNAT in plant development. AtNMNAT catalyzed the synthesis of nicotinate adenine dinucleotide (NaAD) from nicotinate mononucleotide (NaMN) in the Preiss-Handler-dependent pathway, and of nicotinamide adenine dinucleotide (NAD) from nicotiamide mononucleotide (NMN) in the Preiss-Handler-independent pathway. Prominent AtNMNAT expression was detected in the male gametophyte. Moreover, AtNMNAT expression was spatio-temporally regulated during microspore development and pollen tube growth. Disruption of the AtNMNAT gene (atnmnat mutant) was characterized by a decrease in NAD content in pollen. Cytological examinations revealed that the atnmnat mutant was gametophytically impaired in in vivo and in vitro pollen tube growth. Our results suggest that metabolic fulfillment via the NAD pathway is indispensable for normal pollen growth and subsequent normal seed production.     

Nicotinamide adenine dinucleotide metabolism in Candida albicans.

The functional pathways of nicotinamide adenine dinucleotide (NAD) biosynthesis and their regulation were studied in the dimorphic fungus Candida albicans. The presence of a functional endogenous pathway of NAD biosynthesis from tryptophan was demonstrated. In addition, nicotinamide served as an efficient salvage precursor for NAD biosynthesis but nicotinate was not utilized. The pathway for nicotinamide utilization involved nicotinate and nicotinate nucleotides as intermediates, suggesting that the failure to utilize nicotinate involves a transport defect. The mechanisms that regulate NAD levels during exponential growth operated to maintain constant NAD levels when NAD biosynthesis occurred exclusively from endogenous or salvage pathways or from a combination of the two. The regulation also operated such that the salvage pathway was preferentially utilized.     

The pathway of biosynthesis of nicotinamide–adenine dinucleotide in rat mammary gland

1. The pathway of NAD synthesis in mammary gland was examined by measuring the activities of some of the key enzymes in each of the tryptophan, nicotinic acid and nicotinamide pathways. 2. In the tryptophan pathway, 3-hydroxyanthranilate oxidase and quinolinate transphosphoribosylase activities were investigated. Neither of these enzymes was found in mammary gland. 3. In the nicotinic acid pathway, nicotinate mononucleotide pyrophosphorylase, NAD synthetase, nicotinamide deamidase and NMN deamidase were investigated. Both NAD synthetase and nicotinate mononucleotide pyrophosphorylase were present but were very inactive. Nicotinamide deamidase, if present, had a very low activity and NMN deamidase was absent. 4. In the nicotinamide pathway both enzymes, NMN pyrophosphorylase and NMN adenylyltransferase, were present and showed very high activity. The activity of the pyrophosphorylase in mammary gland is by far the highest yet found in any tissue. 5. The apparent K(m) values for the substrates of these enzymes in mammary gland were determined. 6. On the basis of these investigations it is proposed that the main, and probably only, pathway of synthesis of NAD in mammary tissue is from nicotinamide via NMN.     

Isolation and partial characterization of a lectin from a false brome grass (Brachypodium sylvaticum).

Purified rat renal brush-border membrane vesicles possess a heat-labile enzyme activity which hydrolyses NAD+. A reciprocal relationship exists between the disappearance of NAD+ and the appearance of adenosine; 2 mol of Pi are liberated from each mol of NAD+ incubated with brush-border membrane vesicles. Freezing and thawing brush-border membrane vesicles does not enhance the initial rate of NAD+ hydrolysis. Preincubation of brush-border membrane vesicles with NAD+ results in inhibition of Na+-dependent Pi-transport activity, whereas Na+-dependent glucose transport is not affected. EDTA, which prevents the release of Pi from NAD+ and which itself has no direct effect on brush-border membrane Pi transport, reverses the NAD+ inhibition of Na+-dependent Pi transport. These results suggest that it is the Pi liberated from NAD+ and not NAD+ itself that inhibits Na+-dependent Pi transport.     

Importance of nicotinamide as an NAD precursor in the human erythrocyte

The effect of variation in the concentration of inorganic phosphate and of the pyridine precursors nicotinamide (NAm) and nicotinic acid (NA) on pyridine nucleotide synthesis was studied using intact human erythrocytes. A wide range of incubation times was employed. The results showed that under physiological conditions the rate of synthesis of NAD from NAm exceeded that from NA twofold, while the reverse situation pertained at higher and unphysiological substrate levels. The two pathways had different regulation points. For NAm the rate-limiting factor was the initial step, namely its conversion into the mononucleotide, while for NA it lay at the second step, conversion of NA mononucleotide (NAMN) to its adenine dinucleotide. At physiological substrate levels the uptake of NA and conversion to NAMN were rapid, while the uptake and conversion of NAm were time dependent. This process was stimulated significantly by inorganic phosphate only for NAm. These results indicate that while NA is the predominant precursor of human erythrocyte NAD at high (unphysiological) substrate and phosphate levels, NAm is more efficient as an NAD precursor under physiological conditions, suggesting an important and hitherto unrecognized role for nicotinamide in NAD synthesis in vivo.     

Evidence that NADP+ is the physiological cofactor of ADP-L-glycero-D-mannoheptose 6-epimerase

ADP-L-glycero-D-mannoheptose 6-epimerase is required for lipopolysaccharide inner core biosynthesis in several genera of Gram-negative bacteria. The enzyme contains both fingerprint sequences Gly-X-Gly-X-X-Gly and Gly-X-X-Gly-X-X-Gly near its N terminus, which is indicative of an ADP binding fold. Previous studies of this ADP-l-glycero-D-mannoheptose 6-epimerase (ADP-hep 6-epimerase) were consistent with an NAD(+) cofactor. However, the crystal structure of this ADP-hep 6-epimerase showed bound NADP (Deacon, A. M., Ni, Y. S., Coleman, W. G., Jr., and Ealick, S. E. (2000) Structure 5, 453-462). In present studies, apo-ADP-hep 6-epimerase was reconstituted with NAD(+), NADP(+), and FAD. In this report we provide data that shows NAD(+) and NADP(+) both restored enzymatic activity, but FAD could not. Furthermore, ADP-hep 6-epimerase exhibited a preference for binding of NADP(+) over NAD(+). The K(d) value for NADP(+) was 26 microm whereas that for NAD(+) was 45 microm. Ultraviolet circular dichroism spectra showed that apo-ADP-hep 6-epimerase reconstituted with NADP(+) had more secondary structure than apo-ADP-hep 6-epimerase reconstituted with NAD(+). Perchloric acid extracts of the purified enzyme were assayed with NAD(+)-specific alcohol dehydrogenase and NADP(+)-specific isocitric dehydrogenase. A sample of the same perchloric acid extract was analyzed in chromatographic studies, which demonstrated that ADP-hep 6-epimerase binds NADP(+) in vivo. A structural comparison of ADP-hep 6-epimerase with UDP-galactose 4-epimerase, which utilizes an NAD(+) cofactor, has identified the regions of ADP-hep 6-epimerase, which defines its specificity for NADP(+).     

The stoichiometry of the tightly bound NAD+ in urocanase. Separation and characterization of fully active and inhibited forms of the enzyme

1. Urocanase, purified by classical methods [Keul, V., Kaeppeli, F., Ghosh, C., Krebs, T., Robinson, J. A. and Rétey, J. (1979) J. Biol. Chem. 254, 843-851] from Pseudomonas putida was submitted to high-performance liquid chromatography on a TSK-DEAE column. The enzyme was eluted in three resolved peaks (A, B and C) exhibiting specific activities of 3.4 U/mg, 1.85 U/mg and 0.4 U/mg, respectively. 2. The difference spectra of peaks B and A as well as of C and A showed maxima at 330 nm. 3. Irradiation of peaks B and C at 320 nm resulted in an increase of urocanase activity by 45% and 400%, respectively. Peak A could not be photoactivated. Rechromatography of the photoactivated peaks B and C on the TSK-DEAE column confirmed their partial transformation into peak A. 4. Spectroscopic methods for quantitative protein determination were adapted to urocanase. The stoichiometry of bound NAD+/urocanase (form A) was determined to be 1.75 by enzymic analysis of the free NAD+ released upon acid denaturation of the holoenzyme. A similar stoichiometry (1.8-1.9) was found for all three forms (A, B and C) by biosynthetic incorporation of [7-14C]nicotinate into urocanase using a nicotinate auxotrophic mutant of P. putida. 5. Form A of urocanase showed, after treatment with NaBH4 up to 50% inhibition, an elution pattern (TSK-DEAE column) similar to a mixture of forms A, B and C in the approximate ratio of 1:2:1. None of these forms could be photoactivated. 6. We conclude that form A of the urocanase dimer contains two intact NAD+ molecules. In form B one of the two subunits contains an NAD+-nucleophile adduct which is present in both subunits of form C. Full urocanase activity requires intact NAD+ in both subunits. Intact NAD+ can be regenerated from the adduct but not from the reduced form by photolysis. The two subunits of urocanase are independent both in their catalytic activity and in modification reactions.     

Identification of cyclic intermediates in Azorhizobium caulinodans nicotinate catabolism.

In wild-type Azorhizobium caulinodans ORS571, nicotinate served both as anabolic substrate for NAD+ production and as catabolic substrate for use as the N source. Catabolic enzyme activities were greatest from cultures grown with nicotinate as the N source and least when cultures were grown with ammonium as the N source. Vector insertion mutants unable to catabolize nicotinate (nic::Vi mutants) still required micromolar quantities of this compound for growth. Therefore, A. caulinodans wild type is NAD+ auxotrophic. As the first two intermediates in A. caulinodans nicotinate catabolism, two cyclic compounds, 6-hydroxynicotinate and 1,4,5,6-tetrahydro-6-oxonicotinate, were identified. These compounds were purified from the growth medium of strain 61009 (a nic::Vi mutant) by high-performance liquid chromatography; their identities were subsequently confirmed by UV absorbance, nuclear magnetic resonance, and mass spectra. The conversion of 1 mol of nicotinate to 6-hydroxynicotinate consumed 0.5 mol of O2. From 18O isotopic incorporation experiments, water was the hydroxyl-equivalent source. A nicotinate hydroxylase activity proved to be cell wall-membrane associated; this activity served as direct electron donor (not indirect via NADP+) to O2 via membrane electron transport. These catabolic reactions have not previously been witnessed together in the same organism. A. caulinodans nicotinate catabolism seems coupled to N2 fixation, although the explicit mechanism of this coupling remains to be determined.     

Pyridine nucleotide synthesis in 3T3 cells.

The biosynthesis of NAD has been examined in 3T3 cells. The net synthesis of pyridine nucleotides does not occur when cells are cultured in the absence of performed pyridine ring compounds; however, growth continues normally for up to four cell doublings resulting in cells with a total pyridine nucleotide content that is reduced by as much as 12-fold. The mechanism that adjust the relative amounts of NADP and NAD are also altered such that the amount of NADP relative to NAD increases 5-fold. Both nicotinate and nicotinamide can be used as a precursor for NAD biosynthesis, however nicotinate is utilized less efficiently than nicotinamide. The presence of functional pathways for the biosynthesis of NAD from nicotinate via nicotinate mononucleotide and nicotinate adenine dinucleotide and from nicotinamide via nicotinamide mononucleotide has been demonstrated by identification of biosynthetic intermediates following short term exposure of cells to radiolabelled precursors. When cells are grown in Dulbecco's modified Eagle's medium which contains 33 μM nicotinamide the biosynthesis of NAD proceeds by a single pathway with nicotinamide mononucleotide as the only intermediate. Nicotinamide ribonucleoside which previously has been postulated to be an intermediate in the conversion of nicotinamide to NAD is not an intermediate in NAD biosynthesis.     

Comparative aspects of some bacterial dehydrogenases and transhydrogenases

Ragland, T. E. (Brandeis University, Waltham, Mass.), T. Kawasaki, and J. M. Lowenstein. Comparative aspects of some bacterial dehydrogenases and transhydrogenases. J. Bacteriol. 91:236-244. 1966.-Twenty-eight diverse bacterial species were surveyed for the activities and coenzyme specificities of four enzymes: isocitrate dehydrogenase (ICDH), glucose-6-phosphate dehydrogenase (G-6-PDH), 6-phosphogluconate dehydrogenase (6-PGDH), and reduced nicotinamide adenine dinucleotide phosphate-nicotinamide adenine dinucleotide (NAD) transhydrogenase (TH). Most of the species that exhibited a nicotinamide adenine dinucleotide phosphate (NADP)-linked ICDH also showed significant TH activity, but there were several which did not. Only one of the organisms tested, Xanthomonas pruni, had an ICDH active with both NAD and NADP; it was devoid of TH activity. Acetobacter suboxydans, which lacks ICDH altogether, also had no TH. Some of the species examined had G-6-PDH or 6-PGDH (or both) of dual coenzyme specificity, but there was no apparent relation between these findings and the presence or absence of TH. The TH reaction was assayed by use of analogues of NAD as acceptors. The bacteria could be divided into two groups on the basis of TH specificity, one group reacting at a much faster rate with the 3-acetylpyridine analogue of NAD than with the thionicotinamide analogue, whereas the converse was true for the other group. A few organisms showed no marked specificity for either analogue. This division of specificity can be related to the currently accepted taxonomic classification of the organisms, although a few apparent anomalies were found.     

Structural determinants of discrimination of NAD+ from NADH in yeast mitochondrial NADH kinase Pos5

NAD kinase catalyzes the phosphorylation of NAD⁺ to synthesize NADP⁺, whereas NADH kinase catalyzes conversion of NADH to NADPH. The mitochondrial protein Pos5 of Saccharomyces cerevisiae shows much higher NADH kinase than NAD kinase activity and is therefore referred to as NADH kinase. To clarify the structural determinant underlying the high NADH kinase activity of Pos5 and its selectivity for NADH over NAD⁺, we determined the tertiary structure of Pos5 complexed with NADH at a resolution of 2.0 Å. Detailed analysis, including a comparison of the tertiary structure of Pos5 with the structures of human and bacterial NAD kinases, revealed that Arg-293 of Pos5, corresponding to His-351 of human NAD kinase, confers a positive charge on the surface of NADH-binding site, whereas the corresponding His residue does not. Accordingly, conversion of the Arg-293 into a His residue reduced the ratio of NADH kinase activity to NAD kinase activity from 8.6 to 2.1. Conversely, simultaneous changes of Ala-330 and His-351 of human NAD kinase into Ser and Arg residues significantly increased the ratio of NADH kinase activity to NAD kinase activity from 0.043 to 1.39; human Ala-330 corresponds to Pos5 Ser-272, which interacts with the side chain of Arg-293. Arg-293 and Ser-272 were highly conserved in Pos5 homologs (putative NADH kinases), but not in putative NAD kinases. Thus, Arg-293 of Pos5 is a major determinant of NADH selectivity. Moreover, Ser-272 appears to assist Arg-293 in achieving the appropriate conformation.     

Effect of arginine modification on kidney brush-border-membrane transport activity.

The effect of phenylglyoxylation on brush-border-membrane functions was studied with membrane vesicles from rat kidney cortex. Na+-gradient-dependent uptake of phosphate, glucose and alanine was inhibited by 65, 88 and 70% by pre-incubation of vesicles with 50 mM-phenylglyoxal for 2 min. The inhibition showed a dependency for alkaline pH. Borate co-operativity in butanedione inactivation was used to prove that inhibition was caused by arginine modification. Intravesicular volumes, alkaline phosphatase, aminopeptidase M and Na+-H+ exchange were not affected by phenylglyoxal treatment. Inhibition of phosphate uptake was studied in more detail and showed that the chemical modification introduced by phenylglyoxal inhibited the overshoot of phosphate uptake caused by the Na+ gradient, and decreased the apparent maximal velocity of the phosphate-transport system in its interaction with Na+. Phosphate uptake measured in the absence of Na+ was not affected by phenylglyoxal. Shunting of the transmembrane electrical potential with K+ and valinomycin had no effect on phenylglyoxal inhibition, proving that the alteration of transmembrane electrical potential could not be responsible for this effect. Phenylglyoxal had no ionophoric effect on the Na+ gradients studied (1-100 mM). Na+ efflux was also unaffected by phenylglyoxal treatment. Na+, harmaline and amiloride were ineffective in protecting against phenylglyoxal inhibition, suggesting that the site modified was not an Na+-binding site. These results indicate the involvement of highly reactive arginine residues in phosphate, glucose and alanine uptake.