Direct demonstration of an adaptive constraint

The role of constraint in adaptive evolution is an open question. Directed evolution of an engineered beta-isopropylmalate dehydrogenase (IMDH), with coenzyme specificity switched from nicotinamide adenine dinucleotide (NAD) to nicotinamide adenine dinucleotide phosphate (NADP), always produces mutants with lower affinities for NADP. This result is the correlated response to selection for relief from inhibition by NADPH (the reduced form of NADP) expected of an adaptive landscape subject to three enzymatic constraints: an upper limit to the rate of maximum turnover (kcat), a correlation in NADP and NADPH affinities, and a trade-off between NAD and NADP usage. Two additional constraints, high intracellular NADPH abundance and the cost of compensatory protein synthesis, have ensured the conserved use of NAD by IMDH throughout evolution. Our results show that selective mechanisms and evolutionary constraints are to be understood in terms of underlying adaptive landscapes.     

Conformational variability of NAD+ in the free and bound states: a nicotinamide sandwich in NAD+ crystals

X-ray analysis of the free-acid crystal form of the coenzyme nicotinamide adenine dinucleotide (NAD+) revealed a conformational difference between the free NAD+ molecule and one bound in enzymes or complexed to Li+ ions. The pyrophosphate group showed asymmetry in the phosphate-oxygen bonds of the phosphate-oxygen-phosphate link; this bond at the nicotinamide side of the link is longer than that at the adenosine side by 0.04 angstrom. The crystal structure showed a novel intermolecular stacking of adenine and water molecules on opposite sides of nicotinamide that gives rise to a nicotinamide sandwich.     

Lactate dehydrogenase isozymes: further kinetic studies at high enzyme concentration

By competition with lactate dehydrogenase (LDH) for nicotinamide adenine dinucleotide (NAD), commonly occurring intracellular proteins, such as glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase, and albumin, can protect LDH-1 and LDH-5 from inhibition and ternary complex formation with NAD and pyruvate. The existence of intracellular proteins that compete with LDH for NAD renders unphysiological a model for estimating the extent of intracellular LDH inhibition based on incubations of only LDH, NAD, and pyruvate.     

Lactic dehydrogenases: subfractionation of isozymes

Electrophoresis in polyacrylamide gel of homogenates of various organs from the mouse yields five major lactic dehydrogenase bands. If the gels are treated with beta-mercaptoethanol, subsequent electrophoresis produces 15 bands which show lactic dehydrogenase activity. This could be explained if one molecule of nicotinamide adenine dinucleotide (coenzyme) is attached to each of the monomeric subunits of lactic dehydrogenase and if mercaptoethanol can remove the coenzyme only from the muscle type. This is consistent with the hypothesis that intact lactic dehydrogenase is a tetramer.     

MOLECULAR HETEROGENEITY OF ENZYMES: MALATE DEHYDROGENASE OF EUGLENA

Experiments with analogs of the coenzyme nicotinamide adenine dinucleotide demonstrate that the molecular forms of malate dehydrogenase in Euglena vary with the nutritional environment. Electrophoretic separations on starch gel show that Euglena grown on autotrophic medium has a malate dehydrogenase which is lacking in Euglena grown on heterotrophic medium.     

Deuterium effects on binding of reduced coenzyme alcohol dehydrogenase isoenzyme EE

Determination of dissociation constants by two different methods yield the following mean values in 20 millimolar phosphate, pH 7.0, 25 degrees C: 0.27 micromolar for reduced nicotinamide adenine dinucleotide (NADH); 0.29 micromolar for NADH with deuterium in the nicotinamide 4-B position (B-NADD); and 0.46 micromolar for NADH with deuterium in the nicotinamide 4-A position (A-NADD). These results indicate that dehydrogenases are capable of recognizing and distinguishing the appropriate hydrogen in the coenzyme already in the initial binding reaction.     

Fumarate reductase in the control of heme biosynthesis

A drug-induced stimulation of heme biosynthesis in mouse liver was accompanied by altered fumarate metabolism. In liver homogenate, fumarate 1,4-C(14) was incorporated, via succinate and succinyl coenzyme A, into heme at an accelerated rate. This pathway of fumarate utilization was inhibited by acetoacetate but not by beta-hydroxybutyrate. Fumarate reduction to succinate required reduced nicotinamide adenine dinucleotide. The enzyme fumarate reductase is suggested as a link between terminal oxidation and cellular control of the heme biosynthetic pathway.     

Microsomal N-demethylation, by a cotton leaf oxidase system, of 3-(4'-chloropENYL)-1, 1-dimethylurea (monuron)

A cotton leaf microsomal oxidase system that N-demethylates 3-(4'-chlorophenyl)-1,1-dimethylurea (monuron) to 3-(4'-chlorophenyl)-1-methylurea has been partially characterized. The enzyme system is associated with a microsomal fraction separated by differential centrifugation and requires molecular oxygen as well as either the reduced form of nicotinamide adenine dinucleotide phosphate or the reduced form of nicotinamide adenine dinucleotide as cofactors.     

In vivo determination of the pyridine nucleotide reduction charge by carbon-13 nuclear magnetic resonance spectroscopy

An intracellular coenzyme has been observed by carbon-13 nuclear magnetic resonance spectroscopy. The pyridine nucleotides in Escherichia coli were specifically labeled with carbon-13 from the biosynthetic precursor, nicotinic acid. The intracellular redox status and metabolic transformations of the pyridine nucleotides were examined under a variety of conditions. A highly reduced nicotinamide adenine dinucleotide pool was observed under anaerobic conditions only in cells that were cultured aerobically on glycerol.     

Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae

Calorie restriction extends life-span in a wide variety of organisms. Although it has been suggested that calorie restriction may work by reducing the levels of reactive oxygen species produced during respiration, the mechanism by which this regimen slows aging is uncertain. Here, we mimicked calorie restriction in yeast by physiological or genetic means and showed a substantial extension in life-span. This extension was not observed in strains mutant for SIR2 (which encodes the silencing protein Sir2p) or NPT1 (a gene in a pathway in the synthesis of NAD, the oxidized form of nicotinamide adenine dinucleotide). These findings suggest that the increased longevity induced by calorie restriction requires the activation of Sir2p by NAD.     

Immunocyte Ca2+ influx system mediated by LTRPC2

We characterized an activation mechanism of the human LTRPC2 protein, a member of the transient receptor potential family of ion channels, and demonstrated that LTRPC2 mediates Ca2+ influx into immunocytes. Intracellular pyrimidine nucleotides, adenosine 5'-diphosphoribose (ADPR), and nicotinamide adenine dinucleotide (NAD), directly activated LTRPC2, which functioned as a Ca2+-permeable nonselective cation channel and enabled Ca2+ influx into cells. This activation was suppressed by intracellular adenosine triphosphate. These results reveal that ADPR and NAD act as intracellular messengers and may have an important role in Ca2+ influx by activating LTRPC2 in immunocytes.     

Increased nuclear NAD biosynthesis and SIRT1 activation prevent axonal degeneration

Axonal degeneration is an active program of self-destruction that is observed in many physiological and pathological settings. In Wallerian degeneration slow (wlds) mice, Wallerian degeneration in response to axonal injury is delayed because of a mutation that results in overexpression of a chimeric protein (Wlds) composed of the ubiquitin assembly protein Ufd2a and the nicotinamide adenine dinucleotide (NAD) biosynthetic enzyme Nmnat1. We demonstrate that increased Nmnat activity is responsible for the axon-sparing activity of the Wlds protein. Furthermore, we demonstrate that SIRT1, a mammalian ortholog of Sir2, is the downstream effector of increased Nmnat activity that leads to axonal protection. These findings suggest that novel therapeutic strategies directed at increasing the supply of NAD and/or Sir2 activation may be effective for treatment of diseases characterized by axonopathy and neurodegeneration.     

New light-sensitive cofactor required for oxidation of succinate by Mycobacterium phlei

Irradiation of the electron transport particles of Mycobacterium phlei with light at a wavelength of 360 manometers resulted in a loss of oxidase activities of succinate and the reduced form of nicotinamide adenine dinucleotide. The lesion in the two pathways caused by irradiation of the particles differs. The succinoxidase pathway was more labile to irradiation than the pathway linked to nicotinamide adenine dinucleotide. Restoration of succinoxidase activity (up to 50 to 60 percent) occurred on addition of a thermostable, water-soluble material obtained from Mycobacterium phlei cells or with an extract of mitochondria from boiled rat liver. Other known cofactors, such as flavine adenine dinucleotide, flavine mononucleotide, benzo- and naphthoquinones, as well as sulfhydryl agents, failed to restore succinoxidase activity after irradiation. Water-soluble material from Mycobacterium phlei appears to function between the flavoprotein and cytochrome b on the succinoxidase pathway. In contrast to the requirements for restoration of the pathway linked to nicotinamide adenine dinucleotide, restoration of succinoxidase does not occur with quinones or other cofactors such as flavine adenine dinucleotide.     

ABAD directly links Abeta to mitochondrial toxicity in Alzheimer's disease

Mitochondrial dysfunction is a hallmark of beta-amyloid (Abeta)-induced neuronal toxicity in Alzheimer's disease (AD). Here, we demonstrate that Abeta-binding alcohol dehydrogenase (ABAD) is a direct molecular link from Abeta to mitochondrial toxicity. Abeta interacts with ABAD in the mitochondria of AD patients and transgenic mice. The crystal structure of Abeta-bound ABAD shows substantial deformation of the active site that prevents nicotinamide adenine dinucleotide (NAD) binding. An ABAD peptide specifically inhibits ABAD-Abeta interaction and suppresses Abeta-induced apoptosis and free-radical generation in neurons. Transgenic mice overexpressing ABAD in an Abeta-rich environment manifest exaggerated neuronal oxidative stress and impaired memory. These data suggest that the ABAD-Abeta interaction may be a therapeutic target in AD.     

Structural basis for the activation of cholera toxin by human ARF6-GTP

The Vibrio cholerae bacterium causes devastating diarrhea when it infects the human intestine. The key event is adenosine diphosphate (ADP)-ribosylation of the human signaling protein GSalpha, catalyzed by the cholera toxin A1 subunit (CTA1). This reaction is allosterically activated by human ADP-ribosylation factors (ARFs), a family of essential and ubiquitous G proteins. Crystal structures of a CTA1:ARF6-GTP (guanosine triphosphate) complex reveal that binding of the human activator elicits dramatic changes in CTA1 loop regions that allow nicotinamide adenine dinucleotide (NAD+) to bind to the active site. The extensive toxin:ARF-GTP interface surface mimics ARF-GTP recognition of normal cellular protein partners, which suggests that the toxin has evolved to exploit promiscuous binding properties of ARFs.     

A specific, highly active malate dehydrogenase by redesign of a lactate dehydrogenase framework

Three variations to the structure of the nicotinamide adenine dinucleotide (NAD)-dependent L-lactate dehydrogenase from Bacillus stearothermophilus were made to try to change the substrate specificity from lactate to malate: Asp197----Asn, Thr246----Gly, and Gln102----Arg). Each modification shifts the specificity from lactate to malate, although only the last (Gln102----Arg) provides an effective and highly specific catalyst for the new substrate. This synthetic enzyme has a ratio of catalytic rate (kcat) to Michaelis constant (Km) for oxaloacetate of 4.2 x 10(6)M-1 s-1, equal to that of native lactate dehydrogenase for its natural substrate, pyruvate, and a maximum velocity (250 s-1), which is double that reported for a natural malate dehydrogenase from B. stearothermophilus.     

Biochemical modeling of an autonomously oscillatory circadian clock in Euglena

Eukaryotic microorganisms, as well as higher animals and plants, display many autonomous physiological and biochemical rhythmicities having periods approximating 24 hours. In an attempt to determine the nature of the timing mechanisms that are responsible for these circadian periodicities, two primary operational assumptions were postulated. Both the perturbation of a putative element of a circadian clock within its normal oscillatory range and the direct activation as well as the inhibition of such an element should yield a phase shift of an overt rhythm generated by the underlying oscillator. Results of experiments conducted in the flagellate Euglena suggest that nicotinamide adenine dinucleotide (NAD+), the mitochondrial Ca2+-transport system, Ca2+, calmodulin, NAD+ kinase, and NADP+ phosphatase represent clock "gears" that, in ensemble, might constitute a self-sustained circadian oscillating loop in this and other organisms.     

HST2 mediates SIR2-independent life-span extension by calorie restriction

Calorie restriction (CR) extends the life span of numerous species, from yeast to rodents. Yeast Sir2 is a nicotinamide adenine dinucleotide (NAD+-dependent histone deacetylase that has been proposed to mediate the effects of CR. However, this hypothesis has been challenged by the observation that CR can extend yeast life span in the absence of Sir2. Here, we show that Sir2-independent life-span extension is mediated by Hst2, a Sir2 homolog that promotes the stability of repetitive ribosomal DNA, the same mechanism by which Sir2 extends life span. These findings demonstrate that the maintenance of DNA stability is critical for yeast life-span extension by CR and suggest that, in higher organisms, multiple members of the Sir2 family may regulate life span in response to diet.