Nucleotide Moiety Research Articles

Characterization and investigation of substrate specificity of the sugar aminotransferase WecE from E. coli K12.

WecE gene, encoding a sugar aminotransferase (SAT), has been cloned from E. coli K12 and expressed in E. coli BL21 (DE3). The enzyme was purified and characterized. WecE used TDP-4-keto-6-deoxy-D-glucose (TDP-D-Glc4O) and L-glutamate as a good amino acceptor and donor, respectively, leading to the production of TDP-4-amino-4,6-dideoxy-D-galactose (TDP-Fuc4N), which was identified by NMR studies. WecE also showed a similar activity for TDP-4-keto 6-deoxy-D-mannose (TDP-D-Man4O), but no activity for GDP-4-keto-6-deoxy-D-mannose (GDP-D-Man4O), suggesting that the nucleotide moiety would become a key determinant to the substrate specificity of amine acceptor for the activity of the SAT. Multiple alignments showed that SATs have four highly conserved motifs located around the active site and could be divided into three subgroups (VIalpha, VIbeta, and VIgamma) that might be closely related with their substrate specificities.

Sodium chloride-induced self-assembly of microfibers from nanofiber components.

Self-assembly of a nucleotide bolaamphiphile, terminated with two nucleotide moieties including thymine, deoxyribose, and phosphodiester at both ends of the C20 oligomethylene chain, in sodium chloride aqueous solutions has been studied by SEM, LSM, FE-SEM, micro-FTIR, and zeta-potential measurement. The self-assembly behavior of the nucleotide bolaamphiphile was strongly dependent on the concentration of sodium chloride added. The nucleotide bolaamphiphile was found to hierarchically self-assemble to form micrometer-sized fibers (microfibers) consisting of bundles of entangled nanometer-sized fibers (nanofibers) under certain conditions of sodium chloride concentration (approximately 44 mM). The zeta-potential measurement suggested that the surface-charge tuning of the nanofibers induce the hierarchical self-assembly of the microfibers.

From biological analysis to synthetic biology

From biological analysis to synthetic biology

The Cofactor of Tetrachloroethene Reductive Dehalogenase of Dehalospirillum multivorans Is Norpseudo‐B12, a New Type of a Natural Corrinoid

AbstractThe corrinoid cofactor of the tetrachloroethene reductive dehalogenase of Dehalospirillum multivorans was isolated in its Coβ‐cyano form. This cofactor represents the main corrinoid found in D. multivorans cells. Analysis of the isolated cyano‐corrinoid by a combination of HPLC and UV/VIS‐absorbance spectroscopy revealed it to be nonidentical to a variety of known natural B12 derivatives. From high‐resolution mass‐spectrometric analysis, the molecular formula of the corrinoid isolated from D. multivorans could be deduced as C58H81CoN17O14P. The sample of the novel corrinoid from D. multivorans was further analyzed by UV/VIS, CD, and one‐ and two‐dimensional 1H‐, 13C‐, and 15N‐NMR spectroscopy, which indicated its structure to be closely related to that of pseudovitamin B12 (Coβ‐cyano‐7″‐adeninylcobamide). By the same means, the corrinoid could be shown to differ from pseudovitamin B12 only by the lack of the methyl group attached to carbon 176, and, therefore, it was named norpseudovitamin B12 (or, more precisely, 176‐norpseudovitamin B12). Norpseudovitamin B12 represents the first example of a ‘complete’ B12‐cofactor that lacks one of the methyl groups of the cobamide moiety, indicating that the B12‐biosynthetic pathway in D. multivorans differs from that of other organisms. X‐Ray crystal‐structures were determined for norpseudovitamin B12 from D. multivorans and the analogues pseudovitamin B12 and factor A (Coβ‐cyano‐7″‐[2‐methyl]adeninylcobamide). These first accurate crystal structures of complete corrinoids with an adeninyl pseudonucleotide confirmed the expected coordination properties around Co and corroborated the close conformational similarity of the nucleotide moieties of norpseudovitamin B12 and its two homologues.

Insight into the Role of Escherichia coli MobB in Molybdenum Cofactor Biosynthesis Based on the High Resolution Crystal Structure

Two proteins, which are co-transcribed in Escherichia coli (MobA and MobB), are involved in the attachment of a nucleotide moiety to the molybdenum cofactor to form active molybdopterin guanine dinucleotide. Although not essential for this process, the dimeric MobB increases the activation of molybdoenzymes, incorporating this cofactor by a mechanism that is not understood. The structure of MobB has been elucidated in two crystal forms, one of which has provided a model at 1.9-A resolution with Rwork and Rfree values of 21.5 and 28.7%, respectively. The MobB subunit displays an alpha/beta-fold arranged into a major and minor domain, the latter of which is inserted between the major and minor domains of the partner subunit, creating an elongated dimer constructed around a 16-stranded beta-sheet. Structural homologues have been identified, and they include a number of nucleotide-binding proteins. Comparisons indicate that although the phosphate-binding regions are highly conserved, MobB lacks the elements of structure required to interact with and efficiently bind a nucleotide base. In the present structure, a sulfate is bound to the Walker A phosphate-binding motif of MobB. The possibility that MobB forms a complex with the nucleotide-binding MobA, the protein with which it is co-transcribed, is explored, and modeling suggests that such a MobA:MobB complex is feasible. This hypothesis is supported by recent biochemical evidence indicating that MobB interacts with several proteins involved in various stages of molybdenum cofactor biosynthesis including MobA. We propose therefore that MobB is an adapter protein that acts in concert with MobA to achieve the efficient biosynthesis and utilization of molybdopterin guanine dinucleotide.

The crystal structure of coenzyme B12-dependent glycerol dehydratase in complex with cobalamin and propane-1,2-diol.

Recombinant glycerol dehydratase of Klebsiella pneumoniae was purified to homogeneity. The subunit composition of the enzyme was most probably alpha 2 beta 2 gamma 2. When (R)- and (S)-propane-1,2-diols were used independently as substrates, the rate with the (R)-enantiomer was 2.5 times faster than that with the (S)-isomer. In contrast to diol dehydratase, an isofunctional enzyme, the affinity of the enzyme for the (S)-isomer was essentially the same or only slightly higher than that for the (R)-isomer (Km(R)/Km(S) = 1.5). The crystal structure of glycerol dehydratase in complex with cyanocobalamin and propane-1,2-diol was determined at 2.1 A resolution. The enzyme exists as a dimer of the alpha beta gamma heterotrimer. Cobalamin is bound at the interface between the alpha and beta subunits in the so-called 'base-on' mode with 5,6-dimethylbenzimidazole of the nucleotide moiety coordinating to the cobalt atom. The electron density of the cyano group was almost unobservable, suggesting that the cyanocobalamin was reduced to cob(II)alamin by X-ray irradiation. The active site is in a (beta/alpha)8 barrel that was formed by a central region of the alpha subunit. The substrate propane-1,2-diol and essential cofactor K+ are bound inside the (beta/alpha)8 barrel above the corrin ring of cobalamin. K+ is hepta-coordinated by the two hydroxyls of the substrate and five oxygen atoms from the active-site residues. These structural features are quite similar to those of diol dehydratase. A closer contact between the alpha and beta subunits in glycerol dehydratase may be reminiscent of the higher affinity of the enzyme for adenosylcobalamin than that of diol dehydratase. Although racemic propane-1,2-diol was used for crystallization, the substrate bound to glycerol dehydratase was assigned to the (R)-isomer. This is in clear contrast to diol dehydratase and accounts for the difference between the two enzymes in the susceptibility of suicide inactivation by glycerol.

Kinetic, Raman, NMR, and site-directed mutagenesis studies of the Pseudomonas sp. strain CBS3 4-hydroxybenzoyl-CoA thioesterase active site.

4-Hydroxybenzoyl-coenzyme A (4-HBA-CoA) thioesterase catalyzes the hydrolysis of 4-HBA-CoA to 4-hydroxybenzoate and CoA. X-ray crystallographic analysis of the liganded enzyme has shown that the benzoyl thioester and pantetheine moieties of the substrate ligand are bound in a narrow crevice while the nucleotide moiety rests on the protein surface (Thoden, J. B., Holden, H. M., Zhuang, Z. and Dunaway-Mariano, D. (2002) X-ray Crystallographic Analyses of Inhibitor and Substrate Complexes of Wild-type and Mutant 4-Hydroxybenzoyl-CoA Thioesterase, J. Biol. Chem., in press). Asp17 is positioned in the crevice, close to the substrate thioester C=O, which in turn interacts with the positive pole of an alpha-helix macrodipole. In this paper we report the results from spectral, mutagenesis, and kinetic studies which show (1) that substrate activation involves restricted thioester C=O conformational freedom and a modest enhancement of C=O bond polarization, (2) that the nucleotide unit of the substrate is bound through interaction with the protein surface, and (3) that Asp17 contributes a rate factor of 10(4), consistent with its proposed role of general base or nucleophile.

DNA secondary structure and Raman markers of supercoiling in Escherichia coli plasmid pUC19.

Negative supercoiling in the 2686 bp Escherichia coli plasmid pUC19 is comparable in linking number (Lk(0) = 258) and superhelical density (sigma = -0.05) to the moderate supercoiling exhibited by many eukaryotic chromosomal DNAs in vivo. Supercoiled and relaxed forms of purified pUC19 in aqueous solution (0.1 M NaCl, pH 8.3, 20 degrees C) have been investigated by Raman spectroscopy to assess changes in B-DNA secondary structure induced by superhelical stress and to identify putative Raman markers of DNA supercoiling. We find that supercoiling leads to small but significant changes to the B-form Raman signature of linear DNA. Spectral band shifts in the 780-850 cm(-1) interval are interpreted as resulting from a small net change in the average phosphodiester torsions alpha (O3'-P->-O5'-C5') and zeta (C3'-O3'->-P-O5') from the gauche(-)/gauche(-) range to the gauche(-)/trans range with supercoiling. The magnitude of the spectral intensity change implies that approximately 5% of the nucleotide moieties are affected. Supercoiling also introduces small redistributions of Raman intensity within the 1460-1490 and 1660-1670 cm(-1) intervals, consistent with small structural perturbations. Importantly, no Raman markers of Watson-Crick base pairing, base stacking, or C2'-endo/anti deoxynucleoside conformations are perturbed significantly by supercoiling of pUC19, indicating that the B-DNA structure is largely conserved under moderate superhelical stress. Peak and trough features at 814 and 783 cm(-1), and at 1462 and 1489 cm(-1), respectively, in the Raman difference spectrum between superhelical and relaxed DNA are proposed as markers of moderate negative supercoiling. We also show that in Tris-buffered solutions the Raman signature of supercoiled DNA can be obscured by Raman bands of Tris counterions. The subtle structural perturbations to B-DNA induced by moderate supercoiling are consistent with proposed mechanisms of transcriptional activation.

Role of active site binding interactions in 4-chlorobenzoyl-coenzyme A dehalogenase catalysis.

4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolytic dehalogenation of 4-CBA-CoA to 4-hydroxybenzoyl-CoA (4-HBA-CoA) via a multistep mechanism involving initial attack of Asp145 on C(4) of the substrate benzoyl ring to form a Meisenheimer intermediate (EMc), followed by expulsion of the chloride ion to form an arylated enzyme intermediate (EAr) and then ester hydrolysis in the EAr to form product. This study examines the role of binding interactions in dehalogenase catalysis. The enzyme and substrate groups positioned for favorable binding interaction were identified from the X-ray crystal structure of the enzyme-4-HBA-3'-dephospho-CoA complex. These groups were individually modified (via site-directed mutagenesis or chemical synthesis) for the purpose of disrupting the binding interaction. The changes in the Gibbs free energy of the enzyme-substrate complex (DeltaDeltaG(ES)) and enzyme-transition state complex (DeltaDeltaG) brought about by the modification were measured. Cases where DeltaDeltaG exceeds DeltaDeltaG(ES) are indicative of binding interactions used for catalysis. On the basis of this analysis, we show that the H-bond interactions between the Gly114 and Phe64 backbone amide NHs and the substrate benzoyl C=O group contribute an additional 3.1 kcal/mol of stabilization at the rate-limiting transition state. The binding interactions between the enzyme and the substrate CoA nucleotide moiety also intensify in the rate-limiting transition state, reducing the energy barrier to catalysis by an additional 3.3 kcal/mol. Together, these binding interactions contribute approximately 10(6) to the k(cat)/K(m).

A protein pre-organized to trap the nucleotide moiety of coenzyme B(12): refined solution structure of the B(12)-binding subunit of glutamate mutase from Clostridium tetanomorphum.

Uniformly (13)C,(15)N-labeled MutS, the coenzyme B(12)-binding subunit of glutamate mutase from Clostridium tetanomorphum, was prepared by overexpression from an Escherichia coli strain. Multidimensional heteronuclear NMR spectroscopic experiments with aqueous solutions of (13)C,(15)N-labeled MutS provided signal assignments for roughly 90% of the 1025 hydrogen, 651 carbon, and 173 nitrogen atoms and resulted in about 1800 experimental restraints. Based on the information from the NMR experiments, the structure of MutS was calculated, confirming the earlier, less detailed structure obtained with (15)N-labeled MutS. The refined analysis allowed a precise determination of the secondary and tertiary structure including several crucial side chain interactions. The structures of (the apoprotein) MutS in solution and of the B(12)-binding subunit in the crystal of the corresponding homologous holoenzyme from Clostridium cochlearium differ only in a section that forms the well-structured helix alpha1 in the crystal structure and that also comprises the cobalt-coordinating histidine residue. In the apoprotein MutS, this part of the B(12)-binding subunit is dynamic. The carboxy-terminal end of this section is conformationally flexible and has significant propensity for an alpha-helical structure ("nascent helix"). This dynamic section in MutS is a decisive element for the binding of the nucleotide moiety of coenzyme B(12) and appears to be stabilized as a helix (alpha1) upon trapping of the nucleotide of the B(12) cofactor.

Template nucleotide moieties required for de novo initiation of RNA synthesis by a recombinant viral RNA-dependent RNA polymerase.

The recombinant RNA-dependent RNA polymerase of the bovine viral diarrhea virus specifically requires a cytidylate at the 3' end for the de novo initiation of RNA synthesis (C. C. Kao, A. M. Del Vecchio, and W. Zhong, Virology 253:1-7, 1999). Using RNAs containing nucleotide analogs, we found that the N3 and C4-amino group at the initiation cytidine were required for RNA synthesis. However, the ribose C2'-hydroxyl of the initiating cytidylate can accept several modifications and retain the ability to direct synthesis. The only unacceptable modification is a protonated C2'-amino group. Quite strikingly, the recognition of the functional groups for the initiation cytidylate and other template nucleotides are different. For example, a C5-methyl group in cytidine can direct RNA synthesis at all template positions except at the initiation cytidylate and C2'-amino modifications are tolerated better after the +11 position. When a 4-thiouracil (4sU) base analog that allows only imperfect base pairing with the nascent RNA is placed at different positions in the template, the efficiency of synthesis is correlated with the calculated stability of the template-nascent RNA duplex adjacent to the position of the 4sU. These results define the requirements for the specific interactions required for the initiation of RNA synthesis and will be compared to the mechanisms of initiation by other RNA-dependent and DNA-dependent RNA polymerases.

Multinuclear 1D and 2D NMR Investigations on the Interaction between the Pyrimidic Nucleotides 5′-CMP, 5′-dCMP, and 5′-UMP and Diethyltin Dichloride in Aqueous Medium

The interactions between diethyltin dichloride and 5′-CMP, 5′-dCMP, and 5′-UMP in aqueous solution were investigated by multinuclear 1D and 2D NMR techniques including 119Sn, 15N and 31P nuclei. These studies were combined with electrospray mass spectrometry, infrared spectroscopy, solid state 13C, 31P and 117Sn CP-MAS NMR, and elemental analysis. As demonstrated by 31P-1H HOESY spectroscopy, the diethyltin moiety interacts with the phosphate group of the pyrimidic mononucleotides in the pH range 0.5–3.5. Compound 8 (X = Cl), the solid isolated in this pH range from 5′-CMP, contains two tin atoms bridged by one oxygen and one chlorine atom, each tin atom being linked to the phosphate group of a nucleotide moiety. For 5′-UMP the solid isolated (12) has a dimeric structure with two different tin atoms; it can be formed by dimerization of compound 11 with the elimination of two water molecules. As demonstrated by 1H-119Sn HMQC correlation NMR data and the 2J(119Sn–O–117Sn) coupling constant of 156 Hz, the diethyltin moiety reacts with the O(2′) and O(3′) oxygen atoms of the nucleotides to form a dimeric diethyldioxastannolane at pH > 9.0. Between pH 5.0 and 9.0, no evidence for any interaction between the diethyltin moiety and the nucleotides was found.

Heterologous High Level Expression, Purification, and Enzymological Properties of Recombinant Rat Cobalamin-dependent Methionine Synthase

Rat methionine synthase was expressed chiefly as apoenzyme in recombinant baculovirus-infected insect cells (Yamada, K., Tobimatsu, T., and Toraya, T. (1998) Biosci. Biotech. Biochem. 62, 2155-2160). The apoenzyme produced was very unstable, and therefore, after complexation with methylcobalamin, the functional holoenzyme was purified to homogeneity. The specific activity and apparent K(m) values for substrates were in good agreement with those obtained with purified rat liver enzyme. The electronic spectrum of the purified recombinant enzyme resembled that of cob(II)alamin and changed to a methylcobalamin-like one upon incubation of the enzyme with titanium(III) and S-adenosylmethionine. The rate of oxidative inactivation of the enzyme in the absence of S-adenosylmethionine was slower with a stronger reducing agent like titanium(III). The nucleotide moiety, especially the phosphodiester group, was shown to play an important role in the binding of the coenzyme to apoprotein and thus for catalysis. Upon incubation with the apoenzyme in the absence of a reducing agent, cyano- and aquacobalamin were not effective or were effective only slightly in reconstituting holoenzyme. Ethyl- and propylcobalamin formed inactive complexes with apoenzyme, which were converted to holoenzyme by photolytic activation. Adenosylcobalamin was not able to form a complex with apoenzyme, which was convertible to holoenzyme by photoirradiation.

The Structure of Methylcob(III)alamin in Aqueous Solution - A Water Molecule as Structuring Element of the Nucleotide Loop

The solution structure of methylcob(III)alamin (MeCbl; 3), a natural organometallic corrinoid-B12 cofactor whose crystal structure was published in 1985, was established by NMR-spectroscopic analyses of 3 in aqueous solution. The full set of unambiguously assigned 1H, 13C, and amide 15N signals was consistent with identical constitutional and configurational properties of 3 in solution and in the crystal. Specifically investigated were the conformational characteristics of 3 in solution, in particular of its unique Co-coordinating nucleotide moiety. An extensive set of NOE-derived distance constraints was acquired for this purpose, and of angle constraints, based on three-bond coupling constants. These data were used to calculate the solution structure of 3. Our data revealed that the conformation of the nucleotide loop of 3 differs significantly in aqueous solution and in the crystalline state and indicated the presence of a specific H2O molecule `bound' via cooperative H-bonds to three H-bonding functionalities of the nucleotide loop. The observed conformational differences are attributed to structuring contributors to the nucleotide conformation that differ in solution and in the crystal. Most of these can be assigned to H2O molecules, whose position in the crystal is controlled, in part, by the specific crystal packing.

A new mode of B12 binding and the direct participation of a potassium ion in enzyme catalysis: X-ray structure of diol dehydratase.

Diol dehydratase is an enzyme that catalyzes the adenosylcobalamin (coenzyme B12) dependent conversion of 1,2-diols to the corresponding aldehydes. The reaction initiated by homolytic cleavage of the cobalt-carbon bond of the coenzyme proceeds by a radical mechanism. The enzyme is an alpha2beta2gamma2 heterooligomer and has an absolute requirement for a potassium ion for catalytic activity. The crystal structure analysis of a diol dehydratase-cyanocobalamin complex was carried out in order to help understand the mechanism of action of this enzyme. The three-dimensional structure of diol dehydratase in complex with cyanocobalamin was determined at 2.2 A resolution. The enzyme exists as a dimer of heterotrimers (alphabetagamma)2. The cobalamin molecule is bound between the alpha and beta subunits in the 'base-on' mode, that is, 5,6-dimethylbenzimidazole of the nucleotide moiety coordinates to the cobalt atom in the lower axial position. The alpha subunit includes a (beta/alpha)8 barrel. The substrate, 1,2-propanediol, and an essential potassium ion are deeply buried inside the barrel. The two hydroxyl groups of the substrate coordinate directly to the potassium ion. This is the first crystallographic indication of the 'base-on' mode of cobalamin binding. An unusually long cobalt-base bond seems to favor homolytic cleavage of the cobalt-carbon bond and therefore to favor radical enzyme catalysis. Reactive radical intermediates can be protected from side reactions by spatial isolation inside the barrel. On the basis of unique direct interactions between the potassium ion and the two hydroxyl groups of the substrate, direct participation of a potassium ion in enzyme catalysis is strongly suggested.

The change in the electronic character upon cisplatin binding to guanine nucleotide is transmitted to drive the conformation of the local sugar-phosphate backbone—a quantitative study

The microstructure alteration as a result of cisplatin binding to N7 of guanines in DNA has been herein assessed through the multinuclear temperature-, pH*- and concentration-dependent NMR study of the effect of Pt2+ complexation to 2′-deoxyguanosine 3′,5′-bis(ethyl hydrogen phosphate) 1 and its ribo analogue 3 which mimic the central nucleotide moiety in a trinucleoside diphosphate in the complete absence of intramolecular base–base stacking interactions. The N S pseudorotational equilibrium shifts towards N-type conformers by 17 and 21 percentage points at 298 K, respectively, thereby showing that free energy of platination is transmitted to drive the sugar conformation. The increase in the population of N-type conformers was rationalized with the strengthening of the anomeric effect in both 2′-deoxy and ribo nucleotides upon the formation of the Pt–N7 bond which promotes nO4′ → σ*C1′-N9 orbital interactions due to the reduction of π-electron density in the imidazole part of guanine. The additional stabilization of N-type conformers in Pt2+ complexes of ribonucleotides is due to the tuning of the gauche effect of the [N9–C1′–C2′–O2′] fragment, which is absent in 2′–deoxy-ribo counterparts. The platination of N7 favours N1 deprotonation in 2 and 4 by ΔpKa of 0.7 and 0.9 units in comparison with parent nucleotides 1 and 3, respectively. The N S pseudorotational equilibrium in 1–4 showed classical sigmoidal dependence as a function of pH with pKa-values at the inflection points. The population of S-type conformers has increased upon N1 deprotonation in 1–4 because the anomeric effect weakened due to the increased π-electron density in the imidazole part of the guanine moiety. The formation of the Pt–N7 bond in bifunctional complexes 2 and 4 simultaneously causes a shift of the syn anti equilibrium towards anti by 43 and 63 percentage points, and the increase in the population of εt,N conformers by 20 and 32 percentage points at 278 K, respectively. Only a minor conformational redistribution along β, γ, β+1 and ε–1 torsion angles has been observed, which suggests their weak conformational cooperativity with the N S pseudorotational equilibrium as a result of platination to guanine. In comparison with nucleotide phosphodiesters, apurinic 3′,5′-bis(ethyl hydrogen phosphate) sugars 5 and 6 showed no interaction with Pt2+ and therefore no conformational changes.

The Gene, ialA, Associated with the Invasion of Human Erythrocytes by Bartonella bacilliformis, Designates a Nudix Hydrolase Active on Dinucleoside 5′-Polyphosphates

ialA, one of two genes associated with the invasion of human red blood cells by Bartonella bacilliformis, the causative agent of several diseases, has been cloned and expressed in Escherichia coli. The protein, IalA, contains an amino acid array characteristic of a family of enzymes, the Nudix hydrolases, active on a variety of nucleoside diphosphate derivatives. IalA has been purified, identified, and characterized as an enzyme catalyzing the hydrolysis of members of a class of signaling nucleotides, the dinucleoside polyphosphates, with its highest activity on adenosine 5'-tetraphospho-5'-adenosine (Ap4A), but also hydrolyzing Ap5A, Ap6A, Gp4G, and Gp5G. In each case, a pyrophosphate linkage is cleaved yielding a nucleoside triphosphate and the remaining nucleotide moiety.

EPR spectroscopic evidence for the mechanism-based inactivation of adenosylcobalamin-dependent diol dehydratase by coenzyme analogs.

EPR spectra were measured upon incubation of the complex of diol dehydratase with coenzyme analogs in the presence of 1,2-propanediol, a physiological substrate. When the analog in which the D-ribose moiety of the nucleotide loop was replaced by a trimethylene group was used as coenzyme, essentially the same EPR spectrum as that with adenosylcobalamin was obtained. The higher-field doublet and the lower-field broad signals derived from an organic radical and low-spin Co(II) of cob(II)alamin, respectively, were observed. With the imidazolyl counterpart, base-on cob(II)alamin-like species accumulated, but signals due to an organic radical quickly disappeared. When a coenzyme analog lacking the nucleotide moiety was incubated with apoenzyme in the presence of substrate, the EPR spectrum resembling cob(II)inamide was obtained, but no signals due to an organic radical were observed. From these results, it was concluded that the extinction of organic radical intermediates results in inactivation of the enzyme by these coenzyme analogs. Upon suicide inactivation with a [15N2]imidazolyl analog, the octet signals due to Co(II) showed superhyperfine splitting into doublets, indicating axial coordination of 5,6-dimethylbenzimidazole to the cobalamin bound to diol dehydratase.

Metabolism of uridine 5'-diphosphate-glucose in golgi vesicles from pea stems

Uridine 5'-diphosphate-glucose (UDP-Glc) is transported into the lumen of the Golgi cisternae, where is used for polysaccharide biosynthesis. When Golgi vesicles were incubated with UDP-[3H]Glc, [3H]Glc was rapidly transferred to endogenous acceptors and UDP-Glc was undetectable in Golgi vesicles. This result indicated that a uridine-containing nucleotide was rapidly formed in the Golgi vesicles. Since little is known about the fate of the nucleotide derived from UDP-Glc, we analyzed the metabolism of the nucleotide moiety of UDP-Glc by incubating Golgi vesicles with [alpha-32P]UDP-Glc, [beta-32P]UDP-Glc, and [3H]UDP-Glc and identifying the resulting products. After incubation of Golgi vesicles with these radiolabeled substrates we could detect only uridine 5'-monophosphate (UMP) and inorganic phosphate (Pi). UDP could not be detected, suggesting a rapid hydrolysis of UDP by the Golgi UDPase. The by-products of UDP hydrolysis, UMP and Pi, did not accumulate in the lumen, indicating that they were able to exit the Golgi lumen. The exit of UMP was stimulated by UDP-Glc, suggesting the presence of a putative UDP-Glc/UMP antiporter in the Golgi membrane. However, the exit of Pi was not stimulated by UDP-Glc, suggesting that the exit of Pi occurs via an independent membrane transporter.

Evidence for axial coordination of 5,6-dimethylbenzimidazole to the cobalt atom of adenosylcobalamin bound to diol dehydratase.

It was demonstrated by electron paramagnetic resonance (EPR) spectroscopy that organic radical intermediates disappeared and cob(II)alamin accumulated upon suicide inactivation of diol dehydratase by 2-methyl-1,2-propanediol. The resulting EPR spectra showed that the eight hyperfine lines due to the divalent cobalt atom of cob(II)alamin further split into triplets by the superhyperfine coupling to the 14N nucleus. Essentially the same superhyperfine splitting of the octet into triplets was observed with [14N]- and [15N]apoenzyme. When the adenosyl form of [14N2]- and [15N2]imidazolyl analogues of the coenzyme [Toraya, T., and Ishida, A. (1991) J. Biol. Chem. 266, 5430-5437] was used with unlabeled apoenzyme, the octet showed superhyperfine splitting into triplets and doublets, respectively. Therefore, it was concluded that cobalamin is bound to this enzyme with 5,6-dimethylbenzimidazole coordinating to the cobalt atom. This conclusion is consistent with the fact that the consensus sequence forming part of a cobalamin-binding motif, conserved in methionine synthase and some of the other cobalamin enzymes, was not found in the deduced amino acid sequences of the subunits of diol dehydratase. Adenosylcobinamide methyl phosphate, a coenzyme analogue lacking the nucleotide moiety, underwent cleavage of the cobalt-carbon bond upon binding to the enzyme in the presence of substrate, forming a cob(II)inamide derivative without nitrogenous base coordination, as judged by EPR and optical spectroscopy. Therefore, this analogue may be a useful probe for determining whether the replacement of the 5, 6-dimethylbenzimidazole ligand by a histidine residue takes place upon binding of cobalamin to proteins.