The Streptococcus pyogenes NAD+ glycohydrolase modulates epithelial cell PARylation and HMGB1 release

Streptococcus pyogenes uses the cytolysin streptolysin O (SLO) to translocate an enzyme, the S. pyogenes NAD⁺ glycohydrolase (SPN), into the host cell cytosol. However, the function of SPN in this compartment is not known. As a complication, many S. pyogenes strains express a SPN variant lacking NAD⁺ glycohydrolase (NADase) activity. Here, we show that SPN modifies several SLO‐ and NAD⁺‐dependent host cell responses in patterns that correlate with NADase activity. SLO pore formation results in hyperactivation of the cellular enzyme poly‐ADP‐ribose polymerase‐1 (PARP‐1) and production of polymers of poly‐ADP‐ribose (PAR). However, while SPN NADase activity moderates PARP‐1 activation and blocks accumulation of PAR, these processes continued unabated in the presence of NADase‐inactive SPN. Temporal analyses revealed that while PAR production is initially independent of NADase activity, PAR rapidly disappears in the presence of NADase‐active SPN, host cell ATP is depleted and the pro‐inflammatory mediator high‐mobility group box‐1 (HMGB1) protein is released from the nucleus by a PARP‐1‐dependent mechanism. In contrast, HMGB1 is not released in response to NADase‐inactive SPN and instead the cells release elevated levels of interleukin‐8 and tumour necrosis factor‐α. Thus, SPN and SLO combine to induce cellular responses subsequently influenced by the presence or absence of NADase activity.     

Characterization of Streptococcus pyogenes ��-NAD+ Glycohydrolase: RE-EVALUATION OF ENZYMATIC PROPERTIES ASSOCIATED WITH PATHOGENESIS*

The Gram-positive pathogen Streptococcus pyogenes injects a β-NAD⁺ glycohydrolase (SPN) into the cytosol of an infected host cell using the cytolysin-mediated translocation pathway. In this compartment, SPN accelerates the death of the host cell by an unknown mechanism that may involve its β-NAD⁺-dependent enzyme activities. SPN has been reported to possess the unique characteristic of not only catalyzing hydrolysis of β-NAD⁺, but also carrying out ADP-ribosyl cyclase and ADP-ribosyltransferase activities, making SPN the only β-NAD⁺ glycohydrolase that can catalyze all of these reactions. With the long term goal of understanding how these activities may contribute to pathogenesis, we have further characterized the enzymatic activity of SPN using highly purified recombinant protein. Kinetic studies of the multiple activities of SPN revealed that SPN possessed only β-NAD⁺ hydrolytic activity and lacked detectable ADP-ribosyl cyclase and ADP-ribosyltransferase activities. Similarly, SPN was unable to catalyze cyclic ADPR hydrolysis, and could not catalyze methanolysis or transglycosidation. Kinetic analysis of product inhibition by recombinant SPN demonstrated an ordered uni-bi mechanism, with ADP-ribose being released as a second product. SPN was unaffected by product inhibition using nicotinamide, suggesting that this moiety contributes little to the binding energy of the substrate. Upon transformation, SPN was toxic to Saccharomyces cerevisiae, whereas a glycohydrolase-inactive SPN allowed for viability. Taken together, these data suggest that SPN functions exclusively as a strict β-NAD⁺ glycohydrolase during pathogenesis.     

Identification of essential amino acids in the active center of thyroidal NAD+ glycohydrolase

• 1.1. Purified thyroidal NAD⁺ glycohydrolase has been subjected to the action of a number of group specific reagents in order to gain information concerning its mode of action.
• 2.2. Modification of histidyl residues with diethylpyrocarbonate strongly suppresses the NAD⁺ glycohydrolase activity. Inactivation with this reagent can be reversed to some extent by subsequent treatment with hydroxylamine.
• 3.3. NAD⁺ and ADP-ribose partially protect against inactivation with similar efficiencies.
• 4.4. The incomplete reactivation with hydroxylamine after diethylpyrocarbonate treatment and the selective inactivation by 2,4-pentanedione indicates that apart from one or more essential histidyl residue(s) also lysyl residues are important for activity. NAD⁺ and to a smaller extent ADP-ribose again protect against inactivation by 2,4-pentanedione.
• 5.5. The sensitivity of the enzyme towards N-ethyl-5-phenyl-isooxazolium-3'-sulfonate further points to the importance of carboxylate containing side chains.
• 6.6. The mechanistic implications of these results are discussed.

     

Probing the catalytic mechanism of bovine CD38/NADglycohydrolase by site directed mutagenesis of key active site residues

Bovine CD38/NAD⁺ glycohydrolase catalyzes the hydrolysis of NAD⁺ to nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose via a stepwise reaction mechanism. Our recent crystallographic study of its Michaelis complex and covalently-trapped intermediates provided insights into the modalities of substrate binding and the molecular mechanism of bCD38. The aim of the present work was to determine the precise role of key conserved active site residues (Trp118, Glu138, Asp147, Trp181 and Glu218) by focusing mainly on the cleavage of the nicotinamide–ribosyl bond. We analyzed the kinetic parameters of mutants of these residues which reside within the bCD38 subdomain in the vicinity of the scissile bond of bound NAD⁺. To address the reaction mechanism we also performed chemical rescue experiments with neutral (methanol) and ionic (azide, formate) nucleophiles. The crucial role of Glu218, which orients the substrate for cleavage by interacting with the N-ribosyl 2′-OH group of NAD⁺, was highlighted. This contribution to catalysis accounts for almost half of the reaction energy barrier. Other contributions can be ascribed notably to Glu138 and Asp147 via ground-state destabilization and desolvation in the vicinity of the scissile bond. Key interactions with Trp118 and Trp181 were also proven to stabilize the ribooxocarbenium ion-like transition state. Altogether we propose that, as an alternative to a covalent acylal reaction intermediate with Glu218, catalysis by bCD38 proceeds through the formation of a discrete and transient ribooxocarbenium intermediate which is stabilized within the active site mostly by electrostatic interactions.     

A novel cholesterol‐insensitive mode of membrane binding promotes cytolysin‐mediated translocation by Streptolysin O

Cytolysin‐mediated translocation (CMT), performed by Streptococcus pyogenes, utilizes the cholesterol‐dependent cytolysin Streptolysin O (SLO) to translocate the NAD⁺‐glycohydrolase (SPN) into the host cell during infection. SLO is required for CMT and can accomplish this activity without pore formation, but the details of SLO's interaction with the membrane preceding SPN translocation are unknown. Analysis of binding domain mutants of SLO and binding domain swaps between SLO and homologous cholesterol‐dependent cytolysins revealed that membrane binding by SLO is necessary but not sufficient for CMT, demonstrating a specific requirement for SLO in this process. Despite being the only known receptor for SLO, this membrane interaction does not require cholesterol. Depletion of cholesterol from host membranes and mutation of SLO's cholesterol recognition motif abolished pore formation but did not inhibit membrane binding or CMT. Surprisingly, SLO requires the coexpression and membrane localization of SPN to achieve cholesterol‐insensitive membrane binding; in the absence of SPN, SLO's binding is characteristically cholesterol‐dependent. SPN's membrane localization also requires SLO, suggesting a co‐dependent, cholesterol‐insensitive mechanism of membrane binding occurs, resulting in SPN translocation.     

Asp and Thr are critical for cation exchange mediated by NhaD, Na/H antiporter of Vibrio cholerae

The Vc-NhaD is an Na⁺/H⁺ antiporter from Vibrio cholerae belonging to a new family of bacterial Na⁺/H⁺ antiporters, the NhaD family. In the present work we mutagenized five conserved Asp and Glu residues and one conserved Thr residue to Ala in order to identify amino acids that are critical for the antiport activity. All mutations fall into two distinct groups: (i) four variants, Glu¹⁰⁰Ala, Glu²⁵¹Ala, Glu³⁴²Ala, and Asp³⁹³Ala, did not abolish antiport activity but shifted the pH optimum to more alkaline pH, and (ii) variants Asp³⁴⁴Ala, Asp³⁴⁴Asn, and Thr³⁴⁵Ala caused a complete loss of both Na⁺/H⁺ and Li⁺/H⁺ antiport activity whereas the Asp³⁴⁴Glu variant exhibited reduced Na⁺/H⁺ and Li⁺/H⁺ antiport activity. This is the first mutational analysis of the antiporter of NhaD type and the first demonstration of Thr residue being indispensable for Na⁺/H⁺ antiport. We discuss the possible role of Asp³⁴⁴ and Thr³⁴⁵ in the functioning of Vc-NhaD.     

Disruption of NAD~+ binding site in glyceraldehyde 3-phosphate dehydrogenase affects its intranuclear interactions

AIM:To characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase(GAPDH),and mobility of GAPDH in cancer cells treated with chemotherapeutic agents. METHODS:We used proteomics analysis to detect and characterize phosphorylation sites within human GAPDH. Site-specific mutagenesis and alanine scanning was then performed to evaluate functional significance of phosphorylation sites in the GAPDH polypeptide chain. Enzymatic properties of mutated GAPDH variants were assessed using kinetic studies. Intranuclear dynamics parameters(diffusion coefficient and the immobile fraction) were estimated using fluorescence recovery after photobleaching(FRAP) experiments and confocal microscopy. Molecular modeling experiments were performed to estimate the effects of mutations on NAD+ cofactor binding.RESULTS:Using MALDI-TOF analysis,we identified novel phosphorylation sites within the NAD+ binding center of GAPDH at Y94,S98,and T99. Using polyclonal antibody specific to phospho-T99-containing peptide within GAPDH,we demonstrated accumulation of phospho-T99-GAPDH inthe nuclear fractions of A549,HCT116,and SW48 cancer cel s after cytotoxic stress. We performed site-mutagenesis,and estimated enzymatic properties,intranuclear distribution,and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD+ binding center reduced enzymatic activity of GAPDH due to decreased affinity to NAD+(Km = 741 ± 257 μmol/L in T99 I vs 57 ± 11.1 μmol/L in wild type GAPDH. Molecular modeling experiments revealed the effect of mutations on NAD+ binding with GAPDH. FRAP(fluorescence recovery after photo bleaching) analysis showed that mutations in NAD+ binding center of GAPDH abrogated its intranuclear interactions. CONCLUSION:Our results suggest an important functional role of phosphorylated amino acids in the NAD+ binding center in GAPDH interactions with its intranuclear partners.     

The 1.8 Å cholix toxin crystal structure in complex with NAD+ and evidence for a new kinetic model

Certain Vibrio cholerae strains produce cholix, a potent protein toxin that has diphthamide-specific ADP-ribosyltransferase activity against eukaryotic elongation factor 2. Here we present a 1.8 Å crystal structure of cholix in complex with its natural substrate, nicotinamide adenine dinucleotide (NAD⁺). We also substituted hallmark catalytic residues by site-directed mutagenesis and analyzed both NAD⁺ binding and ADP-ribosyltransferase activity using a fluorescence-based assay. These data are the basis for a new kinetic model of cholix toxin activity. Further, the new structural data serve as a reference for continuing inhibitor development for this toxin class.     

Structures of iron-dependent alcohol dehydrogenase 2 from Zymomonas mobilis ZM4 with and without NAD+ cofactor

The ethanologenic bacterium Zymomonas mobilis ZM4 is of special interest because it has a high ethanol yield. This is made possible by the two alcohol dehydrogenases (ADHs) present in Z. mobilis ZM4 (zmADHs), which shift the equilibrium of the reaction toward the synthesis of ethanol. They are metal-dependent enzymes: zinc for zmADH1 and iron for zmADH2. However, zmADH2 is inactivated by oxygen, thus implicating zmADH2 as the component of the cytosolic respiratory system in Z. mobilis. Here, we show crystal structures of zmADH2 in the form of an apo-enzyme and an NAD⁺-cofactor complex. The overall folding of the monomeric structure is very similar to those of other functionally related ADHs with structural variations around the probable substrate and NAD⁺ cofactor binding region. A dimeric structure is formed by the limited interactions between the two subunits with the bound NAD⁺ at the cleft formed along the domain interface. The catalytic iron ion binds near to the nicotinamide ring of NAD⁺, which is likely to restrict and locate the ethanol to the active site together with the oxidized Cys residue and several nonpolar bulky residues. The structures of the zmADH2 from the proficient ethanologenic bacterium Z. mobilis, with and without NAD⁺ cofactor, and modeling ethanol in the active site imply that there is a typical metal-dependent catalytic mechanism.     

Fate of ecto-NAD+ glycohydrolase during phagocytosis of normal and mannosylated latex beads by murine macrophages

In order to gain a better understanding of the role of ecto-NAD⁺ glycohydrolase, an enzyme predominantly associated with phagocytic cells, we have studied its fate in murine macrophages (splenic, resident peritoneal and Kupffer cells) during phagocytosis of opsonized or mannosylated latex beads. In parallel, we have also monitored nucleotide pyrophosphatase, another ectoenzyme of macrophages. Phagosomes were isolated by flotation in a discontinuous sucrose gradient and the enzyme activities were determined with fluorometric methods. Low levels of NAD⁺ glycohydrolase and nucleotide pyrophosphatase could be measured associated with the phagosomal fractions, eg, respectively less than 4.5% and 10% in spleen macrophages. The phagosomal activities originate from the plasma membrane, ie they were latent and inactivation of ecto-NAD⁺ glycohydrolase with the diazonium salt of sulfanilic acid resulted in a marked decrease of this enzyme activity in the phagosomal fractions. Pre-labelling of the cell surface by [³H]-galactosylation indicated that NAD⁺ glycohydrolase is internalized to a lesser extent than an average surface-membrane unit. These results indicate that if ecto-NAD⁺ glycohydrolase of macrophages can be internalized to a limited extent during phagocytosis of opsonized or mannosylated latex beads, this enzyme appears to be predominantly excluded from the surface area involved in the uptake of such particles.     

Application of linear free-energy relationships to the mechanistic probing of nonenzymatic and NAD+-glycohydrolase-catalyzed hydrolysis of pyridine dinucleotides

The use of NAD⁺ analogs lacking a carbonyl function at position C-3 of the pyridinium moiety allowed the manipulation of the kinetic mechanism of calf spleen NAD⁺ glycohydrolase so as to render the cleavage of the pyridinium-ribose bond rate limiting. The analogs used in this study are relatively poor substrates of the enzyme. They present an affinity for the active site which is independent of the nature of their substituent (K_i = 10 ± 2 μm), suggesting that the specificity of the NAD⁺ glycohydrolase reflects the dynamic steps occurring after the formation of the Michaelis complex. The maximal rates of hydrolysis of the NAD⁺ analogs are very sensitive to the pK_a of the departing pyridine; a Brönsted plot (r = 0.99) gave a β_tg = −0.90 (at 37°C). From this plot we could estimate that for NAD⁺, the specific interaction of the 3-carboxamide group with the active site contributed to the catalysis by decreasing the energy barrier by about 2 kcal mol⁻¹. We have also studied the nonenzymatic hydrolysis of NAD⁺ and its analogs under conditions (pH-independent hydrolysis) which favor a unimolecular mechanism. In this case a linear Brönsted plot was also found (r = 0.99) with β_tg = −1.11 (at 37°C). Our data indicate that NAD⁺ glycohydrolase catalyzes the chemical cleavage of the pyridinium-ribose bond, over a 10³ rate difference, according to a single mechanism involving a late transition state in which the scissile bond is broken. The present study strongly supports our previous hypothesis (F. Schuber, P. Travo, and M. Pascal (1979) Bioorg. Chem. 8, 83) according to which NAD⁺ glycohydrolase catalyses unimolecular decomposition of its substrates with generation of an ADP-ribosyl oxocarbonium ion intermediate which must be stabilized by the active site of the enzyme.     

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.     

Distinct developmental and degenerative functions of SARM1 require NAD+ hydrolase activity

SARM1 is the founding member of the TIR-domain family of NAD⁺ hydrolases and the central executioner of pathological axon degeneration. SARM1-dependent degeneration requires NAD⁺ hydrolysis. Prior to the discovery that SARM1 is an enzyme, SARM1 was studied as a TIR-domain adaptor protein with non-degenerative signaling roles in innate immunity and invertebrate neurodevelopment, including at the Drosophila neuromuscular junction (NMJ). Here we explore whether the NADase activity of SARM1 also contributes to developmental signaling. We developed transgenic Drosophila lines that express SARM1 variants with normal, deficient, and enhanced NADase activity and tested their function in NMJ development. We find that NMJ overgrowth scales with the amount of NADase activity, suggesting an instructive role for NAD⁺ hydrolysis in this developmental signaling pathway. While degenerative and developmental SARM1 signaling share a requirement for NAD⁺ hydrolysis, we demonstrate that these signals use distinct upstream and downstream mechanisms. These results identify SARM1-dependent NAD⁺ hydrolysis as a heretofore unappreciated component of developmental signaling. SARM1 now joins sirtuins and Parps as enzymes that regulate signal transduction pathways via mechanisms that involve NAD⁺ cleavage, greatly expanding the potential scope of SARM1 TIR NADase functions.     

Role of surface residue 184 in the catalytic activity of NADH oxidase from Streptococcus pyogenes

Nicotinamide adenine dinucleotide (NADH) oxidase from Streptococcus pyogenes (SpNox) is a flavoprotein harboring one molecule of noncovalently bound flavin adenine dinucleotide. It catalyzes the oxidation of NADH by reducing molecular O₂ to H₂O directly through a four-electron reduction. In this study, we selected the lysine residues on the surface of SpNox and mutated them into arginine residues to study the effect on the enzyme activity. A single-point mutation (K184R) at the surface of SpNox enhanced NADH oxidase activity by approximately 50 % and improved thermostability with 46.6 % longer half life at 30 °C. Further insights into the function of residue K184 were obtained by substituting it with other nonpolar, polar, positively charged, and negatively charged residues. To elucidate the role of this residue, computer-assisted molecular modeling and substrate docking were performed. The results demonstrate that even a single mutation at the surface of the enzyme induces changes in the interaction at the active site and affects the activity and stability. Additionally, the data also suggest that the K184R mutant can be used as an effective biocatalyst for NAD⁺ regeneration in l-rare sugar production.     

PARP-1 inhibition does not restore oxidant-mediated reduction in SIRT1 activity

Sirtuin1 (SIRT1) deacetylase and poly(ADP-ribose)-polymerase-1 (PARP-1) respond to environmental cues, and both require NAD⁺ cofactor for their enzymatic activities. However, the functional link between environmental/oxidative stress-mediated activation of PARP-1 and SIRT1 through NAD⁺ cofactor availability is not known. We investigated whether NAD⁺ depletion by PARP-1 activation plays a role in environmental stimuli/oxidant-induced reduction in SIRT1 activity. Both H₂O₂ and cigarette smoke (CS) decreased intracellular NAD⁺ levels in vitro in lung epithelial cells and in vivo in lungs of mice exposed to CS. Pharmacological PARP-1 inhibition prevented oxidant-induced NAD⁺ loss and attenuated loss of SIRT1 activity. Oxidants decreased SIRT1 activity in lung epithelial cells; however increasing cellular NAD⁺ cofactor levels by PARP-1 inhibition or NAD⁺ precursors was unable to restore SIRT1 activity. SIRT1 was found to be carbonylated by CS, which was not reversed by PARP-1 inhibition or selective SIRT1 activator. Overall, these data suggest that environmental/oxidant stress-induced SIRT1 down-regulation and PARP-1 activation are independent events despite both enzymes sharing the same cofactor.     

Pocket analysis of the full-length cholix toxin. An assessment of the structure–dynamics of the apo catalytic domain

Cholix toxin from Vibrio cholerae is the third member of the diphtheria toxin (DT) group of mono-ADP-ribosyltransferase (mART) bacterial toxins. It shares structural and functional properties with Pseudomonas aeruginosa exotoxin A and Corynebacterium diphtheriae DT. Cholix toxin is an important model for the development of antivirulence approaches and therapeutics against these toxins from pathogenic bacteria. Herein, we have used the high-resolution X-ray structure of full-length cholix complexed with NAD⁺ to describe the properties of the NAD⁺-binding pocket at the residue level, including the role of crystallographic water molecules in the NAD⁺ substrate interaction. The full-length apo cholix structure is used to describe the putative NAD⁺-binding site(s) and to correlate biochemical with crystallographic data to study the stoichiometry and orientation of bound NAD⁺ molecules. We quantitatively describe the NAD⁺ substrate interactions on a residue basis for the main 22 pocket residues in cholix_f, a glycerol and 5 contact water molecules as part of the recognition surface by the substrate according to the conditions of crystallization. In addition, the dynamic properties of an in silico version of the catalytic domain were investigated in order to understand the lack of electronic density for one of the main flexible loops (R-loop) in the pocket of X-ray complexes. Implications for a rational drug design approach for mART toxins are derived.     

A sulfur-based transport pathway in Cu+-ATPases

Cells regulate copper levels tightly to balance the biogenesis and integrity of copper centers in vital enzymes against toxic levels of copper. P_IB-type Cu⁺-ATPases play a central role in copper homeostasis by catalyzing the selective translocation of Cu⁺ across cellular membranes. Crystal structures of a copper-free Cu⁺-ATPase are available, but the mechanism of Cu⁺ recognition, binding, and translocation remains elusive. Through X-ray absorption spectroscopy, ATPase activity assays, and charge transfer measurements on solid-supported membranes using wild-type and mutant forms of the Legionella pneumophila Cu⁺-ATPase (LpCopA), we identify a sulfur-lined metal transport pathway. Structural analysis indicates that Cu⁺ is bound at a high-affinity transmembrane-binding site in a trigonal-planar coordination with the Cys residues of the conserved CPC motif of transmembrane segment 4 (C382 and C384) and the conserved Met residue of transmembrane segment 6 (M717 of the MXXXS motif). These residues are also essential for transport. Additionally, the studies indicate essential roles of other conserved intramembranous polar residues in facilitating copper binding to the high-affinity site and subsequent release through the exit pathway.     

Nad+-dependent isocitrate dehydrogenase from Arabidopsis thaliana. Characterization of two closely related subunits

Two cDNA clones which appear to encode different subunits of NAD⁺-dependent isocitrate dehydrogenase (IDH; EC 1.1.1.41) were identified by homology searches from the Arabidopsis EST database. These cDNA clones were obtained and sequenced; both encoded full-length messages and displayed 82.7% nucleotide sequence identity over the coding region. The deduced amino acid sequences revealed preprotein lengths of 367 residues, with an amino acid identity of 86.1%. Genomic Southern blot analysis showed distinct single-copy genes for both IDH subunits. Both IDH subunits were expressed as recombinant proteins in Escherichia coli, and polyclonal antibodies were raised to each subunit. The Arabidopsis cDNA clones were expressed in Saccharomyces cerevisiae mutants which were deficient in either one or both of the yeast NAD⁺-dependent IDH subunits. The Arabidopsis cDNA clones failed to complement the yeast mutations; although both IDH-I and IDH-II were expressed at detectable levels, neither protein was imported into the mitochondria.     

Effects of molecular weight of dextran and NAD+ density on coenzyme activity of high molecular weight NAD+ derivative covalently bound to dextran

NAD⁺ has been covalently attached to dextrans having different molecular weights to give various NAD⁺ densities (mol NAD⁺ per mol d-glucosyl residue). The effects of molecular weight of dextran and of NAD⁺ density on the coenzyme activity of the dextran-bound NAD⁺ derivatives were examined for the reactions catalysed by alcohol dehydrogenase (alcohol: NAD⁺ oxidoreductase, EC 1.1.1.1) and lactate dehydrogenase (l-lactate:NAD⁺ oxidoreductase, EC 1.1.1.27). The molecular weight of dextran had little effect on coenzyme activity in the range 10 000 to 500 000. At low NAD⁺ density (<0.05 mol NAD⁺/mol d-glucosyl residue), the coenzyme activities of the derivatives were relatively low, but higher densities had little effect on the activity. Dextran-bound NAD⁺ derivatives were twice as stable as free NAD⁺.