Pyrimidine biosynthesis in rat brain

—Studies on the incorporation of [¹⁴C]NaHCO₃ into both orotic acid and RNA in tissue slices reveal the occurrence of the complete orotate pathway for the de novo biosynthesis of pyrimidines in the rat brain. A comparison of the rates of incorporation of bicarbonate into orotic acid and RNA in tissue slices of brain and liver indicate the brain to be one-fourth to one-half as active as the liver in the de novo biosynthesis of pyrimidines. The results of this study favor the proposal that the adult rat brain can meet its needs for pyrimidines through de novo synthesis and is not dependent upon salvage activity and an extraneural supply of pyrimidines.     

Muscle type-specific responses to NAD+ salvage biosynthesis promote muscle function in Caenorhabditis elegans

Salvage biosynthesis of nicotinamide adenine dinucleotide (NAD⁺) from nicotinamide (NAM) lowers NAM levels and replenishes the critical molecule NAD⁺ after it is hydrolyzed. This pathway is emerging as a regulator of multiple biological processes. Here we probe the contribution of the NAM-NAD⁺ salvage pathway to muscle development and function using Caenorhabditis elegans. C. elegans males with mutations in the nicotinamidase pnc-1, which catalyzes the first step of this NAD⁺ salvage pathway, cannot mate due to a spicule muscle defect. Multiple muscle types are impaired in the hermaphrodites, including body wall muscles, pharyngeal muscles and vulval muscles. An active NAD⁺ salvage pathway is required for optimal function of each muscle cell type. However, we found surprising muscle-cell-type specificity in terms of both the timing and relative sensitivity to perturbation of NAD⁺ production or NAM levels. Active NAD⁺ biosynthesis during development is critical for function of the male spicule protractor muscles during adulthood, but these muscles can surprisingly do without salvage biosynthesis in adulthood under the conditions examined. The body wall muscles require ongoing NAD⁺ salvage biosynthesis both during development and adulthood for maximum function. The vulval muscles do not function in the presence of elevated NAM concentrations, but NAM supplementation is only slightly deleterious to body wall muscles during development or upon acute application in adults. Thus, the pathway plays distinct roles in different tissues. As NAM-NAD⁺ biosynthesis also impacts muscle differentiation in vertebrates, we propose that similar complexities may be found among vertebrate muscle cell types.     

Inhibition of fatty acid synthesis by RMI 14,514 (5-tetradecyloxy-2-furoic acid)

RMI 14,514 strongly inhibited the incorporation of label from [1-¹⁴C]acetyl-CoA into fatty acids by rat liver homogenates. No inhibition was observed when [2-¹⁴C]malonyl-CoA was used as the labeled fatty acid precursor. These results suggest that the drug inhibits $\text{de novo}$ fatty acid biosynthesis at the step mediated by acetyl-CoA carboxylase. The data presented in this communication support earlier reports that RMI 14,514 probablyexerts its hypolipidemic effects by inhibition of fatty acid biosynthesis.     

Molecular Mechanism of Distinct Salt-Dependent Enzyme Activity of Two Halophilic Nucleoside Diphosphate Kinases

Nucleoside diphosphate kinases from haloarchaea Haloarcula quadrata (NDK-q) and H. sinaiiensis (NDK-s) are identical except for one out of 154 residues, i.e., Arg³¹ in NDK-q and Cys³¹ in NDK-s. However, the salt-dependent activity profiles of NDK-q and NDK-s are quite different: the optimal NaCl concentrations of NDK-q and NDK-s are 1 M and 2 M, respectively. We analyzed the relationships of the secondary, tertiary, and quaternary structures and NDK activity of these NDKs at various salt concentrations, and revealed that 1), NDK-q is present as a hexamer under a wide range of salt concentrations (0.2-4 M NaCl), whereas NDK-s is present as a hexamer at an NaCl concentration above 2 M and as a dimer at NaCl concentrations below 1 M; 2), dimeric NDK-s has lower activity than hexameric NDK-s; and 3), dimeric NDK-s has higher helicity than hexameric NDK-s. We also determined the crystal structure of hexameric NDK-q, and revealed that Arg³¹ plays an important role in stabilizing the hexamer. Thus the substitution of Arg (as in NDK-q) to Cys (as in NDK-s) at position 31 destabilizes the hexameric assembly, and causes dissociation to less active dimers at low salt concentrations.     

Biochemical and structural characterisation of dehydroquinate synthase from the New Zealand kiwifruit Actinidia chinensis

One of the novel aspects of kiwifruit is the presence of a high level of quinic acid which contributes to the flavour of the fruit. Quinic acid metabolism intersects with the shikimate pathway, which is responsible for the de novo biosynthesis of primary and secondary aromatic metabolites. The gene encoding the enzyme which catalyses the second step of the shikimate pathway, dehydroquinate synthase (DHQS), from the New Zealand kiwifruit Actinidia chinensis was identified, cloned and expressed. A. chinensis DHQS was activated by divalent metal ions, and was found to require NAD⁺ for catalysis. The protein was crystallised and the structure was solved, revealing a homodimeric protein. Each monomer has a NAD⁺ binding site nestled between the distinct N- and C-terminal domains. In contrast to other microbial DHQSs, which show an open conformation in the absence of active site ligands, A. chinensis DHQS adopts a closed conformation. This is the first report of the structure of a DHQS from a plant source.     

The NAD+ synthesizing enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2) is a p53 downstream target

NAD⁺ metabolism plays key roles not only in energy production but also in diverse cellular physiology. Aberrant NAD⁺ metabolism is considered a hallmark of cancer. Recently, the tumor suppressor p53, a major player in cancer signaling pathways, has been implicated as an important regulator of cellular metabolism. This notion led us to examine whether p53 can regulate NAD⁺ biosynthesis in the cell. Our search resulted in the identification of nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2), a NAD⁺ synthetase, as a novel downstream target gene of p53. We show that NMNAT-2 expression is induced upon DNA damage in a p53-dependent manner. Two putative p53 binding sites were identified within the human NMNAT-2 gene, and both were found to be functional in a p53-dependent manner. Furthermore, knockdown of NMNAT-2 significantly reduces cellular NAD⁺ levels and protects cells from p53-dependent cell death upon DNA damage, suggesting an important functional role of NMNAT-2 in p53-mediated signaling. Our demonstration that p53 modulates cellular NAD⁺ synthesis is congruent with p53’s emerging role as a key regulator of metabolism and related cell fate.     

Redox responses in yeast to acetate as the carbon source

Following a shift to medium with acetate as the carbon source, a parental yeast strain exhibited a transient moderate 20% reduction in total cellular [NAD⁺ + NADH] but showed a ∼10-fold increase in the ratio of [NAD⁺]:[NADH] after 36 h. A mutant strain (idhΔ) lacking the tricarboxylic acid cycle enzyme isocitrate dehydrogenase had 50% higher cellular levels of [NAD⁺ + NADH] relative to the parental strain but exhibited similar changes in cofactor concentrations following a shift to acetate medium, despite an inability to grow on that carbon source; essentially all of the cofactor was in the oxidized form within 36 h. The salvage pathway for NAD(H) biosynthesis was found to be particularly important for viability during early transition of the parental strain to stationary phase in acetate medium. However, oxygen consumption was not affected, suggesting that the NAD(H) produced during this time may support other cellular functions. The idhΔ mutant exhibited increased flux through the salvage pathway in acetate medium but was dependent on the de novo pathway for viability. Long-term chronological lifespans of the parental and idhΔ strains were similar, but viability of the mutant strain was dependent on both pathways for NAD(H) biosynthesis.     

Purification and Characterization of an Opioid Antagonist from a Peptic Digest of Bovine κ-Casein

The hepatotoxicity of orally administered secondary autoxidation products of linoleic acid was investigated, as compared with the administration of a saline solution and linoleic acid as controls. The de novo synthesis of fatty acids was strongly reduced in the secondary products group. The level of NADPH in the liver significantly decreased while that of NADH did not. The activities of glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase apparently decreased. The activities of NAD⁺ kinase and NAD⁺ synthetase decreased and that of NAD ⁺ nucleosidase increased in the secondary products group. Therefore, the depletion of NADPH can be attributed to the inhibition of two metabolic systems (an NADPH-supplemental system, and a synthetic system of NADP and NAD), and resulted in the reduction of lipogenesis in the liver.     

Effects of a HP0859 (rfaD) knockout mutation on lipopolysaccharide structure of Helicobacter pylori 26695 and the bacterial adhesion on AGS cells

Lipopolysaccharide (LPS) is considered as an important virulence factor of Helicobacter pylori, and contributes to infection persistence and disease severity. ADP-l-glycero-d-manno-heptose-6-epimerase is an enzyme essential for LPS synthesis and understanding of its biochemistry is critical for drug development. We cloned one putative ortholog of Escherichia colirfaD, HP0859, from H. pylori 26695. Determination of the native molecular weight of the recombinant HP0859 protein suggests that the protein is likely a hexamer. NADP⁺, instead of NAD⁺, was proved to be the physiological cofactor for HP0859 protein. Circular dichroism spectrum analysis demonstrated that the secondary structure of this protein is significantly altered when the cofactor is removed. We also constructed an HP0859 knockout mutant and examined its phenotypic properties. The HP0859 knockout mutant exhibited a severe truncation of LPS, a decreased growth rate, and a higher susceptibility to novobiocin. Disruption of HP0859 also reduced the adhesive capacity of H. pylori to AGS cells, and the infected cells failed to display the classic hummingbird phenotype. Complementation of the HP0859 knockout mutation restored these phenotypes completely. In conclusion, we demonstrate that HP0859 codes for a protein essential for the LPS inner core biosynthesis in H. pylori and an intact LPS structure contributes to the adherence ability of this bacterium.     

Quinolinic acid phosphoribosyltransferase: purification and partial characterization from human liver and brain

Quinolinic acid phosphoribosyltransferase (QPRT) [EC 2.4.2.19] from human liver and brain was purified to homogeneity. Identity of the pure enzymes isolated from the two organs was proven by biochemical, physiocochemical and, following the production and partial purification of anti-liver QPRT antibodies, immunological techniques. Human QPRT has a molecular weight of 170,000 and consists of five identical subunits. Kinetic analyses revealed a Km of 5.6 microM for the substrate (quinolinic acid) and 23 microM for the co-substrate (phosphoribosylpyrophosphate). Enzyme activity was dependent on Mg2+ (optimal concentration: 1 mM) and was inhibited by the enzymatic by-product, inorganic pyrophosphate. Pure QPRT and its antibodies will constitute useful tools in the examination of the possible role of quinolinic acid in the pathogenesis of human neurodegenerative disorders.     

Structure and activity of the NAD(P)+‐dependent succinate semialdehyde dehydrogenase YneI from Salmonella typhimurium

Aldehyde dehydrogenases are found in all organisms and play an important role in the metabolic conversion and detoxification of endogenous and exogenous aldehydes. Genomes of many organisms including Escherichia coli and Salmonella typhimurium encode two succinate semialdehyde dehydrogenases with low sequence similarity and different cofactor preference (YneI and GabD). Here, we present the crystal structure and biochemical characterization of the NAD(P)⁺‐dependent succinate semialdehyde dehydrogenase YneI from S. typhimurium. This enzyme shows high activity and affinity toward succinate semialdehyde and exhibits substrate inhibition at concentrations of SSA higher than 0.1 mM. YneI can use both NAD⁺ and NADP⁺ as cofactors, although affinity to NAD⁺ is 10 times higher. High resolution crystal structures of YneI were solved in a free state (1.85 Å) and in complex with NAD⁺ (1.90 Å) revealing a two domain protein with the active site located in the interdomain interface. The NAD⁺ molecule is bound in the long channel with its nicotinamide ring positioned close to the side chain of the catalytic Cys268. Site‐directed mutagenesis demonstrated that this residue, as well as the conserved Trp136, Glu365, and Asp426 are important for activity of YneI, and that the conserved Lys160 contributes to the enzyme preference to NAD⁺. Our work has provided further insight into the molecular mechanisms of substrate selectivity and activity of succinate semialdehyde dehydrogenases. © 2012 Wiley Periodicals, Inc.     

Cylindrical hexameric antimony complexes, one with an enclosed hexaaquanickel(II) ion

The reaction of meso-2,3-dimercaptosuccinic acid and antimony trioxide produces an unusual hexameric cylindrical antimony complex. The cavity is large enough to accommodate the hexaaquanickel(II) cation. The crystal structures of both the parent hexamer and the Ni(II) species enclosed in the hexamer are reported. Bond valence sums were used to confirm that the antimony is +3 in both species and that the Ni ions are all +2 in the encapsulated complex.     

Crystallization of NAD+ synthetase from Bacillus subtilis

NAD⁺-synthetase is a ubiquitous enzyme catalyzing the last step in the biosynthesis of NAD⁺. Mutants of NAD⁺ synthetase with impaired cellular functions have been isolated, indicating a key role for this enzyme in cellular metabolism. Crystals of the enzyme from Bacillus subtilis suitable for x-ray crystallographic investigation have been grown from polyethylene glycol solutions. Investigation on the structural organization of NAD⁺ synthetase, an enzyme fundamental for NAD⁺ biosynthesis, and belonging to the recently characterized amidotransferase enzymatic family, will provide more insight into the catalytic mechanism of deamido-NAD⁺ → NAD⁺ conversion, a biosynthetic process that is a potential target for the development of antibiotic compounds against Bacillus sp. and related bacteria. © 1996 Wiley-Liss, Inc.     

Control of tetrapyrrole biosynthesis by alternate quaternary forms of porphobilinogen synthase

Porphobilinogen synthase (PBGS) catalyzes the first common step in the biosynthesis of tetrapyrroles (such as heme and chlorophyll). Although the predominant oligomeric form of this enzyme, as inferred from many crystal structures, is that of a homo-octamer, a rare human PBGS allele, F12L, reveals the presence of a hexameric form. Rearrangement of an N-terminal arm is responsible for this oligomeric switch, which results in profound changes in kinetic behavior. The structural transition between octamer and hexamer must proceed through an unparalleled equilibrium containing two different dimer structures. The allosteric magnesium, present in most PBGS, has a binding site in the octamer but not in the hexamer. The unprecedented structural rearrangement reported here relates to the allosteric regulation of PBGS and suggests that alternative PBGS oligomers may function in a magnesium-dependent regulation of tetrapyrrole biosynthesis in plants and some bacteria.     

alpha-linolenic acid biosynthesis in Cyanidium caldarium.

Although α-linolenic acid is nearly absent from Cyanidium caldarium cultured at 53 °C, it is the most abundant unsaturated fatty acid in 20 °C-grown cells. A sudden growth temperature shift of 55 to 25 °C does not stimulate the immediate biosynthesis of α-linolenic acid. However, after an induction period of 48 h, synthesis of α-linolenic acid from acetate can be detected, and the fatty acid accumulates in phosphatidyl choline and sulfolipid. The newly synthesized α-linolenic acid appears to be formed primarily by de novo synthesis and to a much lesser extent from the elongation of a previously formed hexadecatrienoic acid precursor. On the other hand, when a cell-free algal preparation was presented with a hexadecatrienoic acid precursor in the presence of [¹⁴C] malonyl-CoA, the α-linolenic acid formed demonstrated a synthesis by elongation of the precursor. While the cell appears enzymatically capable of α-linolenic acid biosynthesis by both the de novo and elongation processes, de novo synthesis of α-linolenic acid appears to be the more significant mode of synthesis.     

Inhibitory effects of gallic acid and quercetin on UDP-glucose dehydrogenase activity

We have examined polyphenols as potential inhibitors of UDP-glucose dehydrogenase (UGDH) activity. Gallic acid and quercetin decreased specific activities of UGDH and inhibited the proliferation of MCF-7 human breast cancer cells. Western blot analysis showed that gallic acid and quercetin did not affect UGDH protein expression, suggesting that UGDH activity is inhibited by polyphenols at the post-translational level. Kinetics studies using human UGDH revealed that gallic acid was a non-competitive inhibitor with respect to UDP-glucose and NAD⁺. In contrast, quercetin showed a competitive inhibition and a mixed-type inhibition with respect to UDP-glucose and NAD⁺, respectively. These results indicate that gallic acid and quercetin are effective inhibitors of UGDH that exert strong antiproliferative activity in breast cancer cells.     

Developmental regulation of NAD+ kinase in Neurospora crassa

The specific activity of NAD⁺ kinase (ATP:NAD⁺ 2-phosphotransferase, EC 2.7.1.23) from Neurospora crassa shows sharp peaks when the organism enters a new developmental stage of the asexual life cycle: the peaks are observed during hydration and germination of conidia, at the transition from exponential to stationary growth and at the photostimulated conidiation. As stimulation of NAD⁺ kinase activity by light in conidiating mycelium is not sensitive to translation inhibitors, the activiation of pre-existing molecules, rather than induction of protein synthesis de novo may be supposed. Enzyme electrophoresis revealed the presence of four forms of NAD⁺ kinase having different apparent molecular weights (I=333,000; II=306,000; III=229,000 and IV=203,000). Manifestation of the activity of individual forms of NAD⁺ kinase is developmentally controlled: form III is most abundant during vegetative growth, forms I and II prevail in conidia. At the conidial germination the increase of NAD⁺ kinase activity is associated with the activation of form III, whereas during photostimulation of conidiation form II is the most activated one. Therefore, certain molecular forms of the enzyme may be regarded as biochemical markers for different developmental stages of N. crassa.     

Regulation of NAD<Superscript>+</Superscript>-dependent isocitrate dehydrogenase in the citrate producing yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

The mechanism of the increased accumulation (overproduction) of citric acids in the yeast Yarrowia lipolytica while growing in the presence of glucose under nitrogen deficiency was investigated. The limitation of the yeast growth by the source of nitrogen decreases the total content of nucleotides and increases the ratios of ATP/AMP and NADH/NAD⁺. NAD⁺-Dependent isocitrate dehydrogenase, an enzyme of the tricarboxylic acid cycle playing a key role in the regulation of biosynthesis of citric and isocitric acids, was isolated from Y. lipolytica. The molecular weights of the native enzyme and its subunits were found to be 412 and 52 kD, respectively. It is concluded that the enzyme is a homooligomer consisting of eight subunits. Investigation of the effect of some intermediates of the tricarboxylic acid cycle on the activity of this enzyme suggests that the enhanced excretion of citric acids can be caused by the inhibition of NAD⁺-dependent isocitrate dehydrogenase due to the decrease in the content of AMP and increase in the NADH/NAD⁺ ratio in the cells of Y. lipolytica under depletion of nitrogen.Translated from Biokhimiya, Vol. 69, No. 12, 2004, pp. 1706–1714.Original Russian Text Copyright © 2004 by Morgunov, Solodovnikova, Sharyshev, Kamzolova, Finogenova.