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Mammalian CD38 and its Aplysia homolog, ADP-ribosyl cyclase (cyclase), are two prominent enzymes that catalyze the synthesis and hydrolysis of cyclic ADP-ribose (cADPR), a Ca(2+) messenger molecule responsible for regulating a wide range of cellular functions. Although both use NAD as a substrate, the cyclase produces cADPR, whereas CD38 produces mainly ADP-ribose (ADPR). To elucidate the catalytic differences and the mechanism of cyclizing NAD, the crystal structure of a stable complex of the cyclase with an NAD analog, ribosyl-2'F-2'deoxynicotinamide adenine dinucleotide (ribo-2'-F-NAD), was determined. The results show that the analog was a substrate of the cyclase and that during the reaction, the nicotinamide group was released and a stable intermediate was formed. The terminal ribosyl unit at one end of the intermediate formed a close linkage with the catalytic residue (Glu-179), whereas the adenine ring at the other end stacked closely with Phe-174, suggesting that the latter residue is likely to be responsible for folding the linear substrate so that the two ends can be cyclized. Mutating Phe-174 indeed reduced cADPR production but enhanced ADPR production, converting the cyclase to be more CD38-like. Changing the equivalent residue in CD38, Thr-221 to Phe, correspondingly enhanced cADPR production, and the double mutation, Thr-221 to Phe and Glu-146 to Ala, effectively converted CD38 to a cyclase. This study provides the first detailed evidence of the cyclization process and demonstrates the feasibility of engineering the reactivity of the enzymes by mutation, setting the stage for the development of tools to manipulate cADPR metabolism in vivo.

Cyclic ADP-ribose (cADPR) is a universal calcium messenger molecule that regulates many physiological processes. The production and degradation of cADPR are catalyzed by a family of related enzymes, including the ADP-ribosyl cyclase from Aplysia california (ADPRAC) and CD38 from human. Although ADPRC and CD38 share a common evolutionary ancestor, their enzymatic functions toward NAD and cADPR homeostasis have evolved divergently. Thus, ADPRC can only generate cADPR from NAD (cyclase), whereas CD38, in contrast, has multiple activities, i.e. in cADPR production and degradation, as well as NAD hydrolysis (NADase). In this study, we determined a number of ADPRC and CD38 structures bound with various nucleotides. From these complexes, we elucidated the structural features required for the cyclization (cyclase) reaction of ADPRC and the NADase reaction of CD38. Using the structural approach in combination with site-directed mutagenesis, we identified Phe-174 in ADPRC as a critical residue in directing the folding of the substrate during the cyclization reaction. Thus, a point mutation of Phe-174 to glycine can turn ADPRC from a cyclase toward an NADase. The equivalent residue in CD38, Thr-221, is shown to disfavor the cyclizing folding of the substrate, resulting in NADase being the dominant activity. The comprehensive structural comparison of CD38 and APDRC presented in this study thus provides insights into the structural determinants for the functional evolution from a cyclase to a hydrolase.

Stimulation of Jurkat T cells by high concentrations of concanavalin A (ConA) induced an elevation of the endogenous adenosine diphosphoribose (ADPR) concentration and an inward current significantly different from the Ca2+ release-activated Ca2+ current (I(CRAC)). Electrophysiological characterization and activation of a similar current by infusion of ADPR indicated that the ConA-induced current is carried by TRPM2. Expression of TRPM2 in the plasma membrane of Jurkat T cells was demonstrated by reverse transcription-PCR, Western blot, and immunofluorescence. Inhibition of ADPR formation reduced ConA-mediated, but not store-operated, Ca2+ entry and prevented ConA-induced cell death of Jurkat cells. Moreover, gene silencing of TRPM2 abolished the ADPR- and ConA-mediated inward current. Thus, ADPR is a novel second messenger significantly involved in ConA-mediated cell death in T cells.

Modifications at the 2'-position of the nicotinamide-ribosyl moiety influence dramatically the nature of the interactions of the modified beta-NAD+ with calf spleen NAD+ glycohydrolase (EC 3.2.2.6), an enzyme that cleaves the nicotinamide-ribose bound in NAD(P)+. Nicotinamide arabinoside adenine dinucleotide (ara-NAD+) and nicotinamide 2'-deoxy-2'-fluoroarabinoside adenine dinucleotide (araF-NAD+) are not hydrolyzed at measurable rates and are the first documented examples of reversible slow binding inhibitors of this class of enzyme. The kinetic data obtained are consistent with both slow kon and koff rate constants in the formation of an enzyme-inhibitor complex, i.e. the association rate constants are about 10(4) and 10(6) slower than diffusion rates, respectively, for araF-NAD+ and ara-NAD+, and the half-life of the complex is about 3-10 min for both analogues. The kinetic model does not account for a slow turnover of an ADP-ribosyl-enzyme intermediary complex. AraF-NAD+ is one of the most potent inhibitors described for NAD+ glycohydrolase.

The products of the choleragen-catalyzed reaction of NAD+ with guanidine HCl or L-arginine have been isolated by high performance liquid chromatography. Analysis of 1H NMR spectra obtained at 360 MHz establishes the structure of the isolated products to be adenosine diphosphoribosyl guanidiniums present as 1:1 mixtures of alpha and beta anomeric forms. Direct observation of the choleragen-catalyzed reaction of NAD+ with L-arginine by 1H NMR spectroscopy establishes that choleragen synthesizes an alpha anomeric linkage that is subject to subsequent anomerization. The stereospecificity of the reaction provides further evidence that choleragen behaves as an enzyme and may be related mechanistically to other ADP-ribosyltransferases.

E-5-(2-Bromovinyl)-2'-deoxyuridylate (BrvdUMP), the first metabolite in the processing of the antiviral agent E-5-(2-bromovinyl)-2'-deoxyuridine (BrvdUrd), is an excellent alternate substrate for dTMP synthetase. The nucleophilic catalyst of the enzyme adds to the 6-position of the heterocycle and converts the normally inert 5-bromovinyl group of BrvdUMP to a reactive allylic bromide in which both carbons of the side chain are susceptible to nucleophilic attack. These centers react with nucleophiles in the reaction mixture, 2-mercaptoethanol and water, to give three diastereomeric products which have been isolated and characterized. Possible implications of these findings as related to the mechanism and selectivity of BrvdUrd as an antiviral agent are discussed.

The stereospecificity of the enzyme-dependent oxidation of alpha-[4R-2H]NADH has been determined for four dehydrogenases: two pro-R specific enzymes, pig heart malate dehydrogenase and yeast alcohol dehydrogenase; and two pro-S specific enzymes, rabbit muscle glycerol-3-phosphate dehydrogenase and Rhodopseudomonas spheroides 3-hydroxybutyrate dehydrogenase. In all cases, an enzyme-dependent and substrate-specific oxidation to alpha-NAD+ is observed with the stereochemistry of oxidation identical with that found for the oxidation of the correspondingly labeled beta-NADH. The ability of dehydrogenases from diverse sources to utilize alpha-NADH in a stereochemically competent fashion is discussed in relation to proposed interactions between the nicotinamide sugar moiety and active site residues or obligatory alignments of the pyridine and sugar moieties.

Escherichia coli heat-labile enterotoxin (labile toxin, LT) catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide and the ADP-ribosylation of arginine (Moss, J., and Richardson, S.H. (1978) J. Clin. Invest. 62, 281-285). Analysis of the product of the ADP-ribosylation of arginine by nuclear magnetic resonance spectroscopy indicated that the reaction was stereospecific and resulted in the formation of alpha-ADP-ribosyl-L-arginine. This reaction product rapidly anomerized to yield a mixture of the alpha and beta forms. In the presence of [adenine-U-14C]NAD, E. coli enterotoxin catalyzed the transfer of the radiolabel to proteins; the ADP-ribosylation of proteins was inhibited by arginine methyl ester, an alternative substrate. Digestion of the 14C-protein with snake venom phosphodiesterase released predominantly 5'-AMP. No product was obtained with a mobility similar to that of 2'-(5''-phosphoribosyl)-5'-AMP. This result is consistent with the covalent attachment by the enterotoxin of ADP-ribose rather than poly(ADP-ribose) to protein. Thus, LT is catalytically equivalent to choleragen, an enterotoxin of Vibrio cholerae, and activates adenylate cyclase through a similar stereospecific ADP-ribosylation reaction.

Diphtheria toxin inactivates protein synthesis elongation factor 2 by catalyzing the ADP-ribosylation of a novel derivative of histidine, diphthamide, in the protein (Van Ness, B. G., Howard, J. B., and Bodley, J. W. (1980) J. Biol. Chem. 255, 10710-10716). In this report, we describe experiments involving nuclear Overhauser enhancement NMR spectroscopy which were undertaken to elucidate the site of ADP-ribosylation of diphthamide and the configuration of the glycosidic bond formed by the toxin. The essential result of these experiments is that, in ribosyl-diphthamide obtained by enzymatic digestion of ADP-ribosyl-elongation factor-2, the H-5 imidazole proton is near the R-4 proton of ribose. This result and others are consistent with the interpretation that diphtheria toxin covalently attaches ADP-ribose to the imidazole N-1 of diphthamide via an alpha-glycosidic linkage.

In analogy with bleomycin, deglyco-bleomycin B2 has been found to form a stable, diamagnetic complex with Fe(II) and CO. Although the stoichiometry of this complex appeared to be the same as that formed with bleomycin, the geometry of the deglyco-bleomycin complex was fundamentally different, especially as regards orientation of the beta-aminoalanine moiety. In the presence of Fe(II) and O2, deglyco-bleomycin A2 and deglyco-bleomycin B2 were found to release [3H]thymine from radiolabeled PM-2 DNA; when employed at limiting concentrations, deglyco-bleomycin A2 and B2 gave about half as much [3H]thymine release as the respective bleomycins. In view of the spectral evidence (Burger, R. M., Horwitz, S. B., Peisach, J., and Wittenberg, J. B. (1979) J. Biol. Chem. 254, 12299-12302) that Fe(II) . bleomycin . CO has the same geometry as the complex formed by initial association of bleomycin, Fe(II), and O2, the accumulated data suggest strongly that all metal complexes of bleomycin (derivatives) capable of DNA degradation need not have the same geometry.

Cu(I) and Cu(II) form stable 1:1 complexes with bleomycin (BLM). The affinity of both metals for the drug is greater than that of Fe(II). Cu(I) . BLM A2 binds to calf thymus DNA with about the same affinity as Fe(II) . BLM, as judged by DNA-induced fluorescence quenching of the bithiazole moiety of BLM. Based on 1H NMR and potentiometric titration data, the Cu(I) complexes of BLM are shown to have geometries very different than those of other BLM . metal(II) complexes studied thus far. As Cu(I) . BLM is oxidation-reduction active, its geometry is of importance in defining the structural requirements for BLM activity.

The structure of the prosthetic group of citrate lyase (Klebsiella aerogenes) was studied by nuclear magnetic resonance and mass spectrometry. The spectra at 360 MHz of the nucleoside moiety (2'-ribosyladenosine) show the absence of 2'-hydroxyl proton, thus confirming the 2' position as the site of attachment of the second ribose moiety to the dephospho-CoA. This glycosidic linkage is found to be alpha(1" leads to 2') and is identical to that of poly(ADP-ribose). Studies of permethylation products by mass spectrometry support the above conclusion regarding the location of the ribosidic linkage.

The anticancer drug, bleomycin, causes both single and double strand scission of duplex DNA in vitro, with double strand scission occurring in excess of that expected from the random accumulation of single strand nicks. The mechanism of the preferential double strand scission of DNA by bleomycin has been investigated through the synthesis of a series of double hairpin and linear oligonucleotides designed to contain a single nick-like structure at a defined site to serve as models of bleomycin-damaged duplex DNA. The 3' and/or 5' hydroxyls flanking the nick have been phosphorylated to model the increased negative charge at a bleomycin-generated nick. The ability of bleomycin to cleave the intact strand opposite the nick was then determined by autoradiography. The results demonstrate that phosphorylation at either the 3' or 5' hydroxyl, and especially when both sites are phosphorylated, strongly enhances selective cleavage by bleomycin of the opposite strand. These experiments indicate that bleomycin-mediated double strand scission is a form of self-potentiation in which the high affinity of bleomycin for the initially generated nicked sites leads to a greatly enhanced probability of scission of the strand opposite those sites.