Metabolic function for human renalase: oxidation of isomeric forms of ß-NAD(P)H that are inhibitory to primary metabolism.

Renalase is a recently identified flavoprotein that has been associated with numerous physiological maladies. There remains a prevailing belief that renalase functions as a hormone, imparting an influence on vascular tone and heart rate by oxidizing circulating catecholamines, chiefly epinephrine. This activity, however, has not been convincingly demonstrated in vitro, nor has the stoichiometry of this transformation been shown. In prior work we demonstrated that renalase induced rapid oxidation of low-level contaminants of ß-NAD(P)H solutions ( Beaupre, B. A. et al. (2013) Biochemistry 52 , 8929 - 8937 ; Beaupre, B. A. et al. (2013) J. Am. Chem. Soc . 135 , 13980 - 13987 ). Slow aqueous speciation of ß-NAD(P)H resulted in the production of renalase substrate molecules whose spectrophotometric characteristics and equilibrium fractional accumulation closely matched those reported for α-anomers of NAD(P)H. The fleeting nature of these substrates precluded structural assignment. Here we structurally assign and identify two substrates for renalase. These molecules are 2- and 6-dihydroNAD(P), isomeric forms of ß-NAD(P)H that arise either by nonspecific reduction of ß-NAD(P)(+) or by tautomerization of ß-NAD(P)H (4-dihydroNAD(P)). The pure preparations of these molecules induce rapid reduction of the renalase flavin cofactor (230 s(-1) for 6-dihydroNAD, 850 s(-1) for 2-dihydroNAD) but bind only a few fold more tightly than ß-NADH. We also show that 2- and 6-dihydroNAD(P) are potent inhibitors of primary metabolism dehydrogenases and therefore conclude that the metabolic function of renalase is to oxidize these isomeric NAD(P)H molecules to ß-NAD(P)(+), eliminating the threat they pose to normal respiratory activity.

Study of the cis to trans isomerization of 1-phenyl-2,3-disubstituted tetrahydro-beta-carbolines at C(1). Evidence for the carbocation-mediated mechanism.

The present study was undertaken to shed light on the mechanism of the epimerization of cis-1,2,3-trisubstituted tetrahydro-beta-carbolines into the trans isomers via a potential carbocationic intermediate at C(1). In order to study the pathway involved in C(1)-N(2) bond cleavage, the electronic character of the carbon atom at C-1 was altered by substitution of electron-rich and electron-poor phenyl rings at this position. This provided direct evidence of the effects of charge at the proposed site of the carbocationic intermediate. In this regard, a diverse set of 1-(phenyl substituted)-2-benzyl-3-ethoxycarbonyl-1,2,3,4-tetrahydro-beta-carbolines has been synthesized via the Pictet-Spengler reaction by condensation of l-tryptophan derivatives with electron-poor and electron-rich aromatic aldehydes. The epimers involved in the isomerization mechanism were investigated by dynamic (1)H and (13)C NMR spectroscopic and X-ray crystallographic analyses. The kinetic studies, which involved conversion of cis diastereomers into their trans counterparts, were carried out in dilute TFA/CH(2)Cl(2). The 1-(4-methoxyphenyl) cis diastereomer epimerized at a much faster rate into the corresponding trans diastereomer than the related 1-(4-nitrophenyl) cis diastereomer epimerized. These observations provide support for the carbocationic intermediate in the C(1)-N(2) scission process. The understanding of this epimerization process is of importance when Pictet-Spengler reactions are carried out under acidic conditions during the synthesis of indole alkaloids.

Conformational analysis of the cis- and trans-adducts of the Pictet - Spengler reaction. Evidence for the structural basis for the C(1) - N(2) scission process in the cis- to trans-isomerization.

The stable conformations of both the trans- and cis-1,3-disubstituted Nb-benzyl stereoisomers of the Pictet - Spengler reaction have been determined by NMR spectroscopy and X-ray crystallography in order to better understand the C(1) -N(2) cis- to trans-isomerization process. In the Na-H series, the chair conformation was preferred for the trans-isomer 3a, while the cis-isomer 3b existed predominantly in the boat form. However, in the Na-methyl series (1a, 1b, 2a, 2b), both the cis (1b, 2b) and trans (1a, 2a) diastereomers existed in the chair conformation to relieve the A(1,2)-strain between the Na-methyl function and the substituent at C(1). The difference in the preferred conformations of the cis-isomers in the Na-H and Na-methyl series (as compared to the preferred conformations in the trans-isomers) can be employed to understand the reduced rate of epimerization of cis-2b into trans-2a as compared to 3b into 3a. This provides the structural basis for the carbocation-mediated intermediate in the C(1) - N(2) scission process.

Interaction of Cd2+ with Zn finger 3 of transcription factor IIIA: structures and binding to cognate DNA.

Finger 3 of transcription factor IIIA of Xenopus laevis was synthesized and constituted with Zn(2+) or Cd(2+). The C-block element of the internal control region of the promoter of the 5S rRNA gene binds to the Zn-F3 and Cd-F3 with dissociation constants of 2.6 x 10(-5) and 1.5 x 10(-4) M, respectively. According to NMR spectroscopy, Zn-F3, as well as Cd-F3, exists as a conformational equilibrium that is not susceptible to structural analysis by NMR methods. To restrict the observed conformational flexibility, a mutant F3 (mF3), which differs from F3 in the number and type of amino acids between the cysteine and the histidine ligands, was synthesized. The affinity of Zn-mF3 for the C-block DNA was greatly reduced relative to Zn-F3. Nevertheless, the metal ion dissociation constants of the Zn- and Cd-mF3 complexes remain similar to those of the native structures at 4.5 x 10(-9) and 3.2 x 10(-8) M, respectively. Zn-mF3 is more thermally stable than Cd-mF3, but both adopt similar conformations according to two-dimensional (1)H NMR spectroscopy. Each peptide displays a betabetaalpha fold for its backbone that is typical of this class of zinc finger domains. The(113)Cd ion in (113)Cd-mF3 is coupled to the protons of two cysteine and two histidine residues and characterized by a chemical shift of 567 ppm.

Determination of the stable conformation of GABA(A)-benzodiazepine receptor bivalent ligands by low temperature NMR and X-ray analysis.

The stable conformations of GABA(A)-benzodiazepine receptor bivalent ligands 2 and 3 were determined by low temperature NMR spectroscopy and confirmed by single crystal X-ray analysis. The linear conformation was important for these dimers to access the binding site and exhibit potent in vitro affinity as illustrated for alpha5 subtype selective ligand 2 (15 nM). Bivalent ligand 3 with the 5 atom linker folded back upon itself both in solution and in the solid state, moreover, it did not bind to Bz receptors.

Identification of the internal axial ligand of HO2-cobalt(III)-bleomycin: 1H[15N] HSQC NMR investigation of bleomycin, deglycobleomycin, and their hydroperoxide-cobalt(III) complexes.

The identity of the axial ligand contributed by the drug in hydroperoxide-Co(III)-bleomycin and hydroperoxide-Co(III)-deglycobleomycin has been in doubt. With each structure, a combination of (1)H[(15)N] HSQC and HMBC and (1)H COSY and NOESY NMR spectroscopy was used to observe and completely assign the nonaromatic (15)N chemical shifts of natural abundance bleomycin in the two hydroperoxide-Co(III) structures. Together with the (15)N assignments from a published 1D (15)N spectrum, the results permitted the assignment of the primary amine nitrogen to an axial ligand position in both structures.

Structure of the (113)Cd(3)beta domains from Homarus americanus metallothionein-1: hydrogen bonding and solvent accessibility of sulfur atoms.

The three-dimensional structures of the isolated Cd(3)beta domains from Homarus americanus metallothionein have been determined by NMR methods in order to establish a set of beta-domain structures for comparative analysis. First, it was determined that the Cd-cysteine connectivities forming the Cd(3)S(9) metal center were identical to those observed for the beta(N) domain in the native holoprotein. Time- and temperature-dependence studies of the (113)Cd and (1)H 1D-NMR spectra indicated that the beta(N) domain undergoes slow conformational changes before reaching an equilibrium structure. In addition to structural information provided by the metal-to-cysteine connectivities, Phi, chi(1) and chi(2) angle constraints, three H(N...)S hydrogen bond interactions were also determined from a long-range optimized (1)H(N)-(113)Cd HMQC experiment. A simulated annealing protocol was applied to the distance and angle constraints obtained from the 2D-NMR experiments to calculate the three-dimensional structure of the synthetic Cd(3)beta(N) domain of lobster metallothionein. Structure-reactivity relationships are proposed for the reactions of Cd(3)beta domains with 5,5'-dithiobis(2-nitrobenzoate), based on comparisons of surface exposure of sulfur atoms of the lobster and rabbit Cd(3)beta domain structures. Finally, the surface exposure of the beta domains of lobster is compared with beta domains from mammalian metallothioneins.