Business for ocean sustainability: Early responses of ocean governance in the private sector.

A large sample of 1664 companies-69 directly working in the ocean economy-distributed across 19 industrial sectors was investigated to explore awareness and activation regarding direct and indirect pressures on the ocean, their responses to these pressures, and the disclosure tools used. We examined their accountability and disclosure practices on sustainable development goals (SDGs) using the drivers, pressures, state, welfare, and response accounting framework. Based on their 2019 sustainability reports, just 7% of the companies assessed disclosed on SDG14. However, 51% of these companies can be considered as aware, albeit to varying degrees, of the pressures their industries place on the oceans, 44% deploy mitigating activities, and 26% are aware and actively lead business responses to ocean challenges. Although we have seen just early responses in addressing ocean challenges, companies' awareness and activation must converge to achieve ocean sustainability and move businesses into a truly blue economy.

Crystal structure of human nicotinic acid phosphoribosyltransferase.

Nicotinic acid phosphoribosyltransferase (EC 2.4.2.11) (NaPRTase) is the rate-limiting enzyme in the three-step Preiss-Handler pathway for the biosynthesis of NAD. The enzyme catalyzes the conversion of nicotinic acid (Na) and 5-phosphoribosyl-1-pyrophosphate (PRPP) to nicotinic acid mononucleotide (NaMN) and pyrophosphate (PPi). Several studies have underlined the importance of NaPRTase for NAD homeostasis in mammals, but no crystallographic data are available for this enzyme from higher eukaryotes. Here, we report the crystal structure of human NaPRTase that was solved by molecular replacement at a resolution of 2.9 Å in its ligand-free form. Our structural data allow the assignment of human NaPRTase to the type II phosphoribosyltransferase subfamily and reveal that the enzyme consists of two domains and functions as a dimer with the active site located at the interface of the monomers. The substrate-binding mode was analyzed by molecular docking simulation and provides hints into the catalytic mechanism. Moreover, structural comparison of human NaPRTase with the other two human type II phosphoribosyltransferases involved in NAD biosynthesis, quinolinate phosphoribosyltransferase and nicotinamide phosphoribosyltransferase, reveals that while the three enzymes share a conserved overall structure, a few distinctive structural traits can be identified. In particular, we show that NaPRTase lacks a tunnel that, in nicotinamide phosphoribosiltransferase, represents the binding site of its potent and selective inhibitor FK866, currently used in clinical trials as an antitumoral agent.

Metabolic profiling of alternative NAD biosynthetic routes in mouse tissues.

NAD plays essential redox and non-redox roles in cell biology. In mammals, its de novo and recycling biosynthetic pathways encompass two independent branches, the "amidated" and "deamidated" routes. Here we focused on the indispensable enzymes gating these two routes, i.e. nicotinamide mononucleotide adenylyltransferase (NMNAT), which in mammals comprises three distinct isozymes, and NAD synthetase (NADS). First, we measured the in vitro activity of the enzymes, and the levels of all their substrates and products in a number of tissues from the C57BL/6 mouse. Second, from these data, we derived in vivo estimates of enzymes'rates and quantitative contributions to NAD homeostasis. The NMNAT activity, mainly represented by nuclear NMNAT1, appears to be high and nonrate-limiting in all examined tissues, except in blood. The NADS activity, however, appears rate-limiting in lung and skeletal muscle, where its undetectable levels parallel a relative accumulation of the enzyme's substrate NaAD (nicotinic acid adenine dinucleotide). In all tissues, the amidated NAD route was predominant, displaying highest rates in liver and kidney, and lowest in blood. In contrast, the minor deamidated route showed higher relative proportions in blood and small intestine, and higher absolute values in liver and small intestine. Such results provide the first comprehensive picture of the balance of the two alternative NAD biosynthetic routes in different mammalian tissues under physiological conditions. This fills a gap in the current knowledge of NAD biosynthesis, and provides a crucial information for the study of NAD metabolism and its role in disease.

The enzymology of cytosolic pyrimidine 5'-nucleotidases: functional analysis and physiopathological implications.

In mammals, cellular 5'-nucleotidase (5'-NT) activity (EC 3.1.3.5) encompasses a number of genetically and structurally distinct enzyme forms, either membrane-bound or soluble, mainly cytosolic, that are characterized by broad specificity towards nucleoside 5'-monophosphate substrates differing in base (purine/pyrimidine) and/or sugar (oxy/deoxy-ribose) moieties. In particular, among the cytosolic 5'-NTs active towards pyrimidine nucleotides are cN-III and cdN, ubiquitously distributed in mammalian tissues and treated as a single entity in the early days. cN-III was first linked to a genetic defect , hereditary pyrimidine nucleotidase deficiency, associated to a nonspherocyt ic hemolytic anemia disorder of still unclear mechanism but metabolically characterized by abnormally high levels of pyrimidine compounds and ribonucleoproteins in erythrocytes, as evidenced by occurrence of basophilic stippling on blood smearings. Since the first review on pyrimidine-specific nucleotidases (Amici, A.; Magni, G., Arch. Biochem. Biophys., 2002, 397(2), 184- 190), excellent overviews on the topic appeared in the literature. In the present contribution, the major findings on these two enzymatic proteins, cN-III and cdN, will be described with particular emphasis on the relationships between their structure and function, as well as on their roles in normal and pathological conditions. The catalytic mechanism of both specific hydrolytic and phosphotransferase activities, possessed by both enzymes, will be discussed also in the light of recent solution of both cN-III and cdN three-dimensional structures. This review also focuses on possible therapeutic approaches involving cellular 5'-NTs in detoxifying common antiviral and antineoplastic drugs.

Characterization of human nicotinate phosphoribosyltransferase: Kinetic studies, structure prediction and functional analysis by site-directed mutagenesis.

Nicotinate phosphoribosyltransferase (NaPRT, EC 2.4.2.11) catalyzes the conversion of nicotinate (Na) to nicotinate mononucleotide, the first reaction of the Preiss-Handler pathway for the biosynthesis of NAD(+). Even though NaPRT activity has been described to be responsible for the ability of Na to increase NAD(+) levels in human cells more effectively than nicotinamide (Nam), so far a limited number of studies on the human NaPRT have appeared. Here, extensive characterization of a recombinant human NaPRT is reported. We determined its major kinetic parameters and assayed the influence of different compounds on its enzymatic activity. In particular, ATP showed an apparent dual stimulation/inhibition effect at low/high substrates saturation, respectively, consistent with a negative cooperativity model, whereas inorganic phosphate was found to act as an activator. Among other metabolites assayed, including nucleotides, nucleosides, and intermediates of carbohydrates metabolism, some showed inhibitory properties, i.e. CoA, several acyl-CoAs, glyceraldehyde 3-phosphate, phosphoenolpyruvate, and fructose 1,6-bisphosphate, whereas dihydroxyacetone phosphate and pyruvate exerted a stimulatory effect. Furthermore, in light of the absence of crystallographic data, we performed homology modeling to predict the protein three-dimensional structure, and molecular docking simulations to identify residues involved in the recognition and stabilization of several ligands. Most of these residues resulted universally conserved among NaPRTs, and, in this study, their importance for enzyme activity was validated through site-directed mutagenesis.

Simultaneous single-sample determination of NMNAT isozyme activities in mouse tissues.

A novel assay procedure has been developed to allow simultaneous activity discrimination in crude tissue extracts of the three known mammalian nicotinamide mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1) isozymes. These enzymes catalyse the same key reaction for NAD biosynthesis in different cellular compartments. The present method has been optimized for NMNAT isozymes derived from Mus musculus, a species often used as a model for NAD-biosynthesis-related physiology and disorders, such as peripheral neuropathies. Suitable assay conditions were initially assessed by exploiting the metal-ion dependence of each isozyme recombinantly expressed in bacteria, and further tested after mixing them in vitro. The variable contributions of the three individual isozymes to total NAD synthesis in the complex mixture was calculated by measuring reaction rates under three selected assay conditions, generating three linear simultaneous equations that can be solved by a substitution matrix calculation. Final assay validation was achieved in a tissue extract by comparing the activity and expression levels of individual isozymes, considering their distinctive catalytic efficiencies. Furthermore, considering the key role played by NMNAT activity in preserving axon integrity and physiological function, this assay procedure was applied to both liver and brain extracts from wild-type and Wallerian degeneration slow (Wld(S)) mouse. Wld(S) is a spontaneous mutation causing overexpression of NMNAT1 as a fusion protein, which protects injured axons through a gain-of-function. The results validate our method as a reliable determination of the contributions of the three isozymes to cellular NAD synthesis in different organelles and tissues, and in mutant animals such as Wld(S).

Reducing expression of NAD+ synthesizing enzyme NMNAT1 does not affect the rate of Wallerian degeneration.

NAD(+) synthesizing enzyme NMNAT1 constitutes most of the sequence of neuroprotective protein Wld(S), which delays axon degeneration by 10-fold. NMNAT1 activity is necessary but not sufficient for Wld(S) neuroprotection in mice and 70 amino acids at the N-terminus of Wld(S), derived from polyubiquitination factor Ube4b, enhance axon protection by NMNAT1. NMNAT1 activity can confer neuroprotection when redistributed outside the nucleus or when highly overexpressed in vitro and partially in Drosophila. However, the role of endogenous NMNAT1 in normal axon maintenance and in Wallerian degeneration has not been elucidated yet. To address this question we disrupted the Nmnat1 locus by gene targeting. Homozygous Nmnat1 knockout mice do not survive to birth, indicating that extranuclear NMNAT isoforms cannot compensate for its loss. Heterozygous Nmnat1 knockout mice develop normally and do not show spontaneous neurodegeneration or axon pathology. Wallerian degeneration after sciatic nerve lesion is neither accelerated nor delayed in these mice, consistent with the proposal that other endogenous NMNAT isoforms play a principal role in Wallerian degeneration.

Homology modeling and deletion mutants of human nicotinamide mononucleotide adenylyltransferase isozyme 2: new insights on structure and function relationship.

Nicotinamide mononucleotide adenylyltransferase (NMNAT) catalyzes the formation of NAD by means of nucleophilic attack by 5'-phosphoryl of NMN on the α-phosphoryl group of ATP. Humans possess three NMNAT isozymes (NMNAT1, NMNAT2, and NMNAT3) that differ in size and sequence, gene expression pattern, subcellular localization, oligomeric state and catalytic properties. Of these, NMNAT2, the least abundant isozyme, is the only one whose much-needed crystal structure has not been solved as yet. To fill this gap, we used the crystal structures of human NMNAT1 and NMNAT3 as templates for homology-based structural modeling of NMNAT2, and the resulting raw structure was then refined by molecular dynamics simulations in a water box to obtain a model of the final folded structure. We investigated the importance of NMNAT2's central domain, which we postulated to be dispensable for catalytic activity, instead representing an isozyme-specific control domain within the overall architecture of NMNAT2. Indeed, we experimentally confirmed that removal of different-length fragments from this central domain did not compromise the enzyme's catalytic activity or the overall tridimensional structure of the active site.

Selective inhibition of nicotinamide adenine dinucleotide kinases by dinucleoside disulfide mimics of nicotinamide adenine dinucleotide analogues.

Diadenosine disulfide (5) was reported to inhibit NAD kinase from Listeria monocytogenes and the crystal structure of the enzyme-inhibitor complex has been solved. We have synthesized tiazofurin adenosine disulfide (4) and the disulfide 5, and found that these compounds were moderate inhibitors of human NAD kinase (IC(50)=110 microM and IC(50)=87 microM, respectively) and Mycobacterium tuberculosis NAD kinase (IC(50)=80 microM and IC(50)=45 microM, respectively). We also found that NAD mimics with a short disulfide (-S-S-) moiety were able to bind in the folded (compact) conformation but not in the common extended conformation, which requires the presence of a longer pyrophosphate (-O-P-O-P-O-) linkage. Since majority of NAD-dependent enzymes bind NAD in the extended conformation, selective inhibition of NAD kinases by disulfide analogues has been observed. Introduction of bromine at the C8 of the adenine ring restricted the adenosine moiety of diadenosine disulfides to the syn conformation making it even more compact. The 8-bromoadenosine adenosine disulfide (14) and its di(8-bromoadenosine) analogue (15) were found to be the most potent inhibitors of human (IC(50)=6 microM) and mycobacterium NAD kinase (IC(50)=14-19 microM reported so far. None of the disulfide analogues showed inhibition of lactate-, and inosine monophosphate-dehydrogenase (IMPDH), enzymes that bind NAD in the extended conformation.

Nicotinamide mononucleotide synthetase is the key enzyme for an alternative route of NAD biosynthesis in Francisella tularensis.

Enzymes involved in the last 2 steps of nicotinamide adenine dinucleotide (NAD) cofactor biosynthesis, which catalyze the adenylylation of the nicotinic acid mononucleotide (NaMN) precursor to nicotinic acid dinucleotide (NaAD) followed by its amidation to NAD, constitute promising drug targets for the development of new antibiotics. These enzymes, NaMN adenylyltransferase (gene nadD) and NAD synthetase (gene nadE), respectively, are indispensable and conserved in nearly all bacterial pathogens. However, a comparative genome analysis of Francisella tularensis allowed us to predict the existence of an alternative route of NAD synthesis in this category A priority pathogen, the causative agent of tularaemia. In this route, the amidation of NaMN to nicotinamide mononucleotide (NMN) occurs before the adenylylation reaction, which converts this alternative intermediate to the NAD cofactor. The first step is catalyzed by NMN synthetase, which was identified and characterized in this study. A crystal structure of this enzyme, a divergent member of the NadE family, was solved at 1.9-A resolution in complex with reaction products, providing a rationale for its unusual substrate preference for NaMN over NaAD. The second step is performed by NMN adenylyltransferase of the NadM family. Here, we report validation of the predicted route (NaMN --> NMN --> NAD) in F. tularensis including mathematical modeling, in vitro reconstitution, and in vivo metabolite analysis in comparison with a canonical route (NaMN --> NaAD --> NAD) of NAD biosynthesis as represented by another deadly bacterial pathogen, Bacillus anthracis.

Wld S protein requires Nmnat activity and a short N-terminal sequence to protect axons in mice.

The slow Wallerian degeneration (Wld(S)) protein protects injured axons from degeneration. This unusual chimeric protein fuses a 70-amino acid N-terminal sequence from the Ube4b multiubiquitination factor with the nicotinamide adenine dinucleotide-synthesizing enzyme nicotinamide mononucleotide adenylyl transferase 1. The requirement for these components and the mechanism of Wld(S)-mediated neuroprotection remain highly controversial. The Ube4b domain is necessary for the protective phenotype in mice, but precisely which sequence is essential and why are unclear. Binding to the AAA adenosine triphosphatase valosin-containing protein (VCP)/p97 is the only known biochemical property of the Ube4b domain. Using an in vivo approach, we show that removing the VCP-binding sequence abolishes axon protection. Replacing the Wld(S) VCP-binding domain with an alternative ataxin-3-derived VCP-binding sequence restores its protective function. Enzyme-dead Wld(S) is unable to delay Wallerian degeneration in mice. Thus, neither domain is effective without the function of the other. Wld(S) requires both of its components to protect axons from degeneration.

Non-nuclear Wld(S) determines its neuroprotective efficacy for axons and synapses in vivo.

Axon degeneration contributes widely to neurodegenerative disease but its regulation is poorly understood. The Wallerian degeneration slow (Wld(S)) protein protects axons dose-dependently in many circumstances but is paradoxically abundant in nuclei. To test the hypothesis that Wld(S) acts within nuclei in vivo, we redistributed it from nucleus to cytoplasm in transgenic mice. Surprisingly, instead of weakening the phenotype as expected, extranuclear Wld(S) significantly enhanced structural and functional preservation of transected distal axons and their synapses. In contrast to native Wld(S) mutants, distal axon stumps remained continuous and ultrastructurally intact up to 7 weeks after injury and motor nerve terminals were robustly preserved even in older mice, remaining functional for 6 d. Moreover, we detect extranuclear Wld(S) for the first time in vivo, and higher axoplasmic levels in transgenic mice with Wld(S) redistribution. Cytoplasmic Wld(S) fractionated predominantly with mitochondria and microsomes. We conclude that Wld(S) can act in one or more non-nuclear compartments to protect axons and synapses, and that molecular changes can enhance its therapeutic potential.

Enzymology of mammalian NAD metabolism in health and disease.

Mounting evidence attests to the paramount importance of the non-redox NAD functions. Indeed, NAD homeostasis is related to the free radicals-mediated production of reactive oxygen species responsible for irreversible cellular damage in infectious disease, diabetes, inflammatory syndromes, neurodegeneration and cancer. Because the cellular redox status depends on both the absolute concentration of pyridine dinucleotides and their respective ratios of oxidized and reduced forms (i.e., NAD/NADH and NADP/NADPH), it is conceivable that an altered regulation of the synthesis and degradation of NAD impairs the cell redox state and likely contributes to the mechanisms underlying the pathogenesis of the above mentioned diseases. Taking into account the recent appearance in the literature of comprehensive reviews covering different aspects of the significance of NAD metabolism, with particular attention to the enzymes involved in NAD cleavage, this monograph includes the most recent results on NAD biosynthesis in mammals and humans. Due to recent findings on nicotinamide riboside as a nutrient, its inclusion under "niacins" is proposed. Here, the enzymes involved in the de novo and reutilization pathways are overviewed.

Bifunctional NMN adenylyltransferase/ADP-ribose pyrophosphatase: structure and function in bacterial NAD metabolism.

Bacterial NadM-Nudix is a bifunctional enzyme containing a nicotinamide mononucleotide (NMN) adenylyltransferase and an ADP-ribose (ADPR) pyrophosphatase domain. While most members of this enzyme family, such as that from a model cyanobacterium Synechocystis sp., are involved primarily in nicotinamide adenine dinucleotide (NAD) salvage/recycling pathways, its close homolog in a category-A biodefense pathogen, Francisella tularensis, likely plays a central role in a recently discovered novel pathway of NAD de novo synthesis. The crystal structures of NadM-Nudix from both species, including their complexes with various ligands and catalytic metal ions, revealed detailed configurations of the substrate binding and catalytic sites in both domains. The structure of the N-terminal NadM domain may be exploited for designing new antitularemia therapeutics. The ADPR binding site in the C-terminal Nudix domain is substantially different from that of Escherichia coli ADPR pyrophosphatase, and is more similar to human NUDT9. The latter observation provided new insights into the ligand binding mode of ADPR-gated Ca2+ channel TRPM2.

Initial-rate kinetics of human NMN-adenylyltransferases: substrate and metal ion specificity, inhibition by products and multisubstrate analogues, and isozyme contributions to NAD+ biosynthesis.

Initial-rate and product inhibition studies revealed distinctive ordered ternary complex kinetic mechanisms, substrate specificities, and metal ion preferences for the three isozymes of human nicotinamide mononucleotide adenylyl-transferase (NMNAT, EC 2.7.7.1). ATP binds before NMN with nuclear isozyme NMNAT1 and Golgi apparatus NMNAT2, but the opposite order is observed with the mitochondrial isozyme NMNAT3. Only the latter utilizes ITP efficiently in place of ATP, and while NMNH conversion to NADH by NMNAT1 and NMNAT3 occurs at similar rates, conversion by NMNAT2 is much slower. These isozymes can also be discriminated by their action on tiazofurin monophosphate (TrMP), a metabolite of the antineoplastic prodrug tiazofurin. Our finding that TrMP is only a substrate with NMNAT1 and NMNAT3 reveals for the first time an organelle selectivity in the metabolism of this important drug. In search of additional ways to discriminate these isozymes, we synthesized and tested the P1-(nicotinamide/nicotinate-riboside-5')-Pn-(adenosine-5') dinucleotides Np3AD, Np4AD, and Nap4AD. In addition to being highly effective inhibitors, these multisubstrate geometric inhibitors gave inhibition patterns that are consistent with the aforementioned isozyme differences in substrate binding order. Distinctive differences in their substrate specificity and metal ion selectivity also permitted us to quantify individual isozyme contributions to NAD+ formation in human cell extracts.

Probing binding requirements of NAD kinase with modified substrate (NAD) analogues.

Synthesis of novel NAD(+) analogues that cannot be phosphorylated by NAD kinase is reported. In these analogues the C2' hydroxyl group of the adenosine moiety was replaced by fluorine in the ribo or arabino configuration (1 and 2, respectively) or was inverted into arabino configuration to give compound 3. Compounds 1 and 2 showed inhibition of human NAD kinase, whereas analogue 3 inhibited both the human and Mycobacterium tuberculosis NAD kinase. An uncharged benzamide adenine dinucleotide (BAD) was found to be the most potent competitive inhibitor (K(i)=90 microM) of the human enzyme reported so far.

Structural and functional properties of NAD kinase, a key enzyme in NADP biosynthesis.

NAD kinase is an essential enzyme, which plays a key role in cellular energy and signal transduction systems. In this report, the recent studies on the features of bacterial and human NAD kinases are summarized. They include detailed kinetic and structural analyses and highlight important differences, which could be exploited for the design of novel selective antimicrobial drugs.

Synthesis and biological evaluation of NAD analogs as human pyridine nucleotide adenylyltransferase inhibitors.

NAD analogs modified at the ribose adenylyl moiety, named N-2'-MeAD and Na-2'-MeAD, were synthesized as ligands of pyridine nucleotide (NMN/NaMN) adenylyltransferase (NMNAT). Both dinucleotides resulted selective inhibitors against human NMNAT-3 isoenzyme.

A novel fold revealed by Mycobacterium tuberculosis NAD kinase, a key allosteric enzyme in NADP biosynthesis.

NAD kinase catalyzes the magnesium-dependent phosphorylation of NAD, representing the sole source of freshly synthesized NADP in all organisms. The enzyme is essential for the growth of the deadly multidrug-resistant pathogen Mycobacterium tuberculosis and is an attractive target for novel antitubercular agents. The crystal structure of NAD kinase has been solved by multiwavelength anomalous dispersion at a resolution of 2.3 A in its T state. Two crystal forms have been obtained revealing either a dimer or a tetramer. The enzyme architecture discloses a novel molecular arrangement, with each subunit consisting of an alpha/beta N-terminal domain and a C-terminal 12-stranded beta sandwich domain, connected by swapped beta strands. The C-terminal domain shows a striking internal approximate 222 symmetry and an unprecedented topology, revealing a novel fold within the family of all beta structures. The catalytic site is located in the long crevice that defines the interface between the domains. The conserved GGDG structural fingerprint of the catalytic site is reminiscent of the related region in 6-phosphofructokinase, supporting the hypothesis that NAD kinase belongs to a newly reported superfamily of kinases.

Characterization of Mycobacterium tuberculosis NAD kinase: functional analysis of the full-length enzyme by site-directed mutagenesis.

NAD kinase is the only known enzyme catalyzing the formation of NADP, a coenzyme implicated in most reductive biosynthetic reactions and in many antioxidant defense systems. Despite its importance, nothing is known regarding its structure or mechanism of catalysis. Mycobacterium tuberculosis NAD kinase has been overexpressed in Escherichia coli and purified to homogeneity. The molecular and kinetic properties of the enzyme resulted in significant differences from those reported by others on a proteolytically degraded form of the protein. Indeed the full-length enzyme displays an allosteric behavior and shows a strict preference for inorganic polyphosphate as the phosphate donor. It is inhibited by the reaction product NADP and by both NADH and NADPH. The mycobacterial enzyme shares with all other known NAD kinases a highly conserved region (spanning residues 189-210), particularly rich in glycines, which differs from the primary sequences of all previously identified nucleotide-binding sites. Alanine-scanning mutagenesis performed on 11 conserved residues within this domain revealed its importance in catalysis. A total of 6 of 11 mutated proteins completely lost the enzymatic activity while retaining the same oligomeric state of the wild-type protein, as demonstrated by gel-filtration analysis. Substitutions of S199 and G208 with alanine rendered enzyme versions with reduced activity. Their kinetic characterization, performed on purified proteins, revealed kinetic parameters toward ATP and polyphosphate similar to those of the wild-type enzyme. On the contrary, when the kinetic analysis was performed by using NAD as the variable substrate, significant differences were observed with respect to both the allosteric behavior and the catalytic efficiency, suggesting that the mutated region is likely involved in NAD binding.