47775-00-8Relevant articles and documents
Emissive Synthetic Cofactors: An Isomorphic, Isofunctional, and Responsive NAD+ Analogue
Rovira, Alexander R.,Fin, Andrea,Tor, Yitzhak
supporting information, p. 15556 - 15559 (2017/11/14)
The synthesis, photophysics, and biochemical utility of a fluorescent NAD+ analogue based on an isothiazolo[4,3-d]pyrimidine core (NtzAD+) are described. Enzymatic reactions, photophysically monitored in real time, show NtzAD+ and NtzADH to be substrates for yeast alcohol dehydrogenase and lactate dehydrogenase, respectively, with reaction rates comparable to that of the native cofactors. A drop in fluorescence is seen as NtzAD+ is converted to NtzADH, reflecting a complementary photophysical behavior to that of the native NAD+/NADH. NtzAD+ and NtzADH serve as substrates for NADase, which selectively cleaves the nicotinamide's glycosidic bond yielding tzADP-ribose. NtzAD+ also serves as a substrate for ribosyl transferases, including human adenosine ribosyl transferase 5 (ART5) and Cholera toxin subunit A (CTA), which hydrolyze the nicotinamide and transfer tzADP-ribose to an arginine analogue, respectively. These reactions can be monitored by fluorescence spectroscopy, in stark contrast to the corresponding processes with the nonemissive NAD+.
Biosynthesis of thiamin thiazole in eukaryotes: Conversion of NAD to an advanced intermediate
Chatterjee, Abhishek,Jurgenson, Christopher T.,Schroeder, Frank C.,Ealick, Steven E.,Begley, Tadhg P.
, p. 2914 - 2922 (2007/10/03)
Thiazole synthase catalyzes the formation of the thiazole moiety of thiamin pyrophosphate. The enzyme from Saccharomyces cerevisiae (THI4) copurifies with a set of strongly bound adenylated metabolites. One of them has been characterized as the ADP adduct of 5-(2-hydroxyethyl)-4-methylthiazole-2- carboxylic acid. Attempts toward yielding active wild-type THI4 by releasing protein-bound metabolites have failed so far. Here, we describe the identification and characterization of two partially active mutants (C204A and H200N) of THI4. Both mutants catalyzed the release of the nicotinamide moiety from NAD to produce ADP-ribose, which was further converted to ADP-ribulose. In the presence of glycine, both the mutants catalyzed the formation of an advanced intermediate. The intermediate was trapped with orthophenylenediamine, yielding a stable quinoxaline derivative, which was characterized by NMR spectroscopy and ESI-MS. These observations confirm NAD as the substrate for THI4 and elucidate the early steps of this unique biosynthesis of the thiazole moiety of thiamin in eukaryotes.
Transition state structure for the hydrolysis of NAD+ catalyzed by diphtheria toxin
Berti, Paul J.,Blanke, Steven R.,Schramm, Vern L.
, p. 12079 - 12088 (2007/10/03)
Diphtheria toxin (DTA) uses NAD+ as an ADP-ribose donor to catalyze the ADP-ribosylation of eukaryotic elongation factor 2. This inhibits protein biosynthesis and ultimately leads to cell death. In the absence of its physiological acceptor, DTA catalyzes the slow hydrolysis of NAD+ to ADP- ribose and nicotinamide, a reaction that can be exploited to measure kinetic isotope effects (KIEs) of isotopically labeled NAD+s. Competitive KIEs were measured by the radiolabel method for NAD+ molecules labeled at the following positions: 1-15N = 1.030 ± 0.004, 1'-14C = 1.034 ± 0.004, (1- 15N, 1'-14C) = 1.062 ± 0.010, 1'-3H = 1.200 ± 0.005, 2'-3H = 1.142 ± 0.005, 4'-3H = 0.990 ± 0.002, 5'-3H = 1.032 ± 0.004, 4'-18O = 0.986 ± 0.003. The ring oxygen, 4'-18O, KIE was also measured by whole molecule mass spectrometry (0.991 ± 0.003) and found to be within experimental error of that measured by the radiolabel technique, giving an overall average of 0.988 ± 0.003. The transition state structure of NAD+ hydrolysis was determined using a structure interpolation method to generate trial transition state structures and bond-energy/bond-order vibrational analysis to predict the KIEs of the trial structures. The predicted KIEs matched the experimental ones for a concerted, highly oxocarbenium ion-like transition state. The residual bond order to the leaving group was 0.02 (bond length = 2.65 A?), while the bond order to the approaching nucleophile was 0.03 (2.46 A?). This is an A(N)D(N) mechanism, with both leaving group and nucleophilic participation in the reaction coordinate. Fitting the transition state structure into the active site cleft of the X-ray crystallographic structure of DTA highlighted the mechanisms of enzymatic stabilization of the transition state. Desolvation of the nicotinamide ring, stabilization of the oxocarbenium ion by apposition of the side chain carboxylate of Glu148 with the anomeric carbon of the ribosyl moiety, and the placement of the substrate phosphate near the positively charged side chain of His21 are all consistent with the transition state features from KIE analysis.
