Ribosyl Moiety Research Articles

Electroauxiliary-Assisted C-C Bond Formations in Lithium Perchlorate / Nitromethane Solution

C-nucleosides have C-C bonds between ribosyl moiety and nucleobase at anomeric carbon. This substitution of covalent bond gives hydrolysis resistance to C-nucleosides. Therefore, C-nucleosides are good candidates for antiviral or antitumor drugs as mimic building blocks of oligonucleotide.To synthesize nucleosides, cation intermediate is used for installing nucleobase on ribosyl moiety. When it comes to generation of cation intermediate, electrochemical methodology is one of the useful tools. In electrochemical method, 1’-arylthioriboses are used as glycosyl donors. They have thioether group as electroauxiliary at anomeric carbon forging O,S-acetal that are activated with anodic oxidation to generate corresponding cation intermediate. Since electrochemically generated cation intermediates have high reactivity enough to react with carbon nucleophiles, this method can be adopted to C-nucleoside synthesis.Previously, electrochemical nucleoside synthesis was reported using cation pool method in combination with electroauxiliary. To avoid undesired oxidation of nucleophile, it is important to pool the cation and/or to lower the oxidation potential of ribose. The low temperature condition such as -78 ~ -50 oC is necessary to pool cation intermediates.In this research, we aimed to achieve electrochemical C-C bond formation at anomeric carbon with ambient temperature condition. In this strategy, we use glycosyl donors with lowered oxidation potential to avoid competitive oxidation with carbon nucleophiles. We also adapt lithium perchlorate/nitromethane system for electrolysis. Figure 1

Analysis of the Relationship between PARP1 and BRCA1 Suggests PARP1 Gene Has a Role in Breast Cancer

Poly [ADP-ribose] polymerase 1 (PARP1), one of the genes in the PARP family, is mainly involved in the detection and repair of DNA damage in cells, and its upregulation has been associated with tumorigenesis. PARP1 is essential to all cells in the body as it ensures that DNA is replicated correctly. The PARP1 protein addresses repair of single-stranded breaks (SSBs) in DNA by initially binding near the point of break in the DNA. While it is bound to the region of the SSB, PARP1 transfers ADP ribosyl moiety from NAD+ to acceptor proteins. This leads to the recruitment of DNA repair proteins to the region of DNA breaks. The PARP1 protein also aids the double-stranded break (DSB) repair of DNA by recruiting the homologous recombination (HR) pathway proteins. Due to this role of PARP1 in DNA repair, it has been associated with cancer growth. Using databases, including UniProt, Xena Browser, and CBioPortal, this article explores the relationship between PARP1 mutations and BRCA1-mutated breast cancer. BRCA1 is a tumor suppressor gene, which is involved in DNA repair through the HR pathway. Therefore, in BRCA1-mutated cells, the lack of tumor suppressor factors leads to cancer growth. However, in a cell that has both BRCA1 mutations and PARP1 inhibition, DNA damage cannot be repaired, leading to apoptosis. Hence, PARP1 inhibitors have become essential in BRCA1-mutated breast cancers. There are several PARP1 inhibitors that are currently being used to treat breast cancer, which work using several mechanisms of action, including PARP1 trapping, which prevents the repair, transcription, and replication of DNA. This article also focuses on the clinical trials of three specific drugs, Olaparib, Iniparib, and Veliparib, which are used to treat breast cancer. However, due to the lack of understanding surrounding PARP1 inhibition mechanisms and potential drug combinations, more research needs to be done to understand potential biomarker targets and PARP1 inhibitor resistance.

Structural similarities between SAM and ATP recognition motifs and detection of ATP binding in a SAM binding DNA methyltransferase

S-adenosylmethionine (SAM) is a ubiquitous co-factor that serves as a donor for methylation reactions and additionally serves as a donor of other functional groups such as amino and ribosyl moieties in a variety of other biochemical reactions. Such versatility in function is enabled by the ability of SAM to be recognized by a wide variety of protein molecules that vary in their sequences and structural folds. To understand what gives rise to specific SAM binding in diverse proteins, we set out to study if there are any structural patterns at their binding sites. A comprehensive analysis of structures of the binding sites of SAM by all-pair comparison and clustering, indicated the presence of 4 different site-types, only one among them being well studied. For each site-type we decipher the common minimum principle involved in SAM recognition by diverse proteins and derive structural motifs that are characteristic of SAM binding. The presence of the structural motifs with precise three-dimensional arrangement of amino acids in SAM sites that appear to have evolved independently, indicates that these are winning arrangements of residues to bring about SAM recognition. Further, we find high similarity between one of the SAM site types and a well known ATP binding site type. We demonstrate using in vitro experiments that a known SAM binding protein, HpyAII.M1, a type 2 methyltransferase can bind and hydrolyse ATP. We find common structural motifs that explain this, further supported through site-directed mutagenesis. Observation of similar motifs for binding two of the most ubiquitous ligands in multiple protein families with diverse sequences and structural folds presents compelling evidence at the molecular level in favour of convergent evolution.

Arginine ADP-Ribosylation: Chemical Synthesis of Post-Translationally Modified Ubiquitin Proteins.

We describe the development and optimization of a methodology to prepare peptides and proteins modified on the arginine residue with an adenosine-di-phosphate-ribosyl (ADPr) group. Our method comprises reacting an ornithine containing polypeptide on-resin with an α-linked anomeric isothiourea N-riboside, ensuing installment of a phosphomonoester at the 5'-hydroxyl of the ribosyl moiety followed by the conversion into the adenosine diphosphate. We use this method to obtain four regioisomers of ADP-ribosylated ubiquitin (UbADPr), each modified with an ADP-ribosyl residue on a different arginine position within the ubiquitin (Ub) protein (Arg42, Arg54, Arg72, and Arg74) as the first reported examples of fully synthetic arginine-linked ADPr-modified proteins. We show the chemically prepared Arg-linked UbADPr to be accepted and processed by Legionella enzymes and compare the entire suite of four Arg-linked UbADPr regioisomers in a variety of biochemical experiments, allowing us to profile the activity and selectivity of Legionella pneumophila ligase and hydrolase enzymes.

DELTEX E3 ligases ubiquitylate ADP-ribosyl modification on protein substrates.

Ubiquitylation had been considered limited to protein lysine residues, but other substrates have recently emerged. Here, we show that DELTEX E3 ligases specifically target the 3' hydroxyl of the adenosine diphosphate (ADP)-ribosyl moiety that can be linked to a protein, thus generating a hybrid ADP-ribosyl-ubiquitin modification. Unlike other known hydroxyl-specific E3s, which proceed via a covalent E3~ubiqutin intermediate, DELTEX enzymes are RING E3s that stimulate a direct ubiquitin transfer from E2~ubiquitin onto a substrate. However, DELTEXes follow a previously unidentified paradigm for RING E3s, whereby the ligase not only forms a scaffold but also provides catalytic residues to activate the acceptor. Comparative analysis of known hydroxyl-ubiquitylating active sites points to the recurring use of a catalytic histidine residue, which, in DELTEX E3s, is potentiated by a glutamate in a catalytic triad-like manner. In addition, we determined the hydrolase specificity profile of this modification, identifying human and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enzymes that could reverse it in cells.

Structural Insights into Binding of Remdesivir Triphosphate within the Replication-Transcription Complex of SARS-CoV-2.

Remdesivir is an adenosine analogue that has a cyano substitution in the C1′ position of the ribosyl moiety and a modified base structure to stabilize the linkage of the base to the C1′ atom with its strong electron-withdrawing cyano group. Within the replication–transcription complex (RTC) of SARS-CoV-2, the RNA-dependent RNA polymerase nsp12 selects remdesivir monophosphate (RMP) over adenosine monophosphate (AMP) for nucleotide incorporation but noticeably slows primer extension after the added RMP of the RNA duplex product is translocated by three base pairs. Cryo-EM structures have been determined for the RTC with RMP at the nucleotide-insertion (i) site or at the i + 1, i + 2, or i + 3 sites after product translocation to provide a structural basis for a delayed-inhibition mechanism by remdesivir. In this study, we applied molecular dynamics (MD) simulations to extend the resolution of structures to the measurable maximum that is intrinsically limited by MD properties of these complexes. Our MD simulations provide (i) a structural basis for nucleotide selectivity of the incoming substrates of remdesivir triphosphate over adenosine triphosphate and of ribonucleotide over deoxyribonucleotide, (ii) new detailed information on hydrogen atoms involved in H-bonding interactions between the enzyme and remdesivir, and (iii) direct information on the catalytically active complex that is not easily captured by experimental methods. Our improved resolution of interatomic interactions at the nucleotide-binding pocket between remedesivir and the polymerase could help to design a new class of anti-SARS-CoV-2 inhibitors.

Synthesis of Azido and Triazolyl Purine Ribonucleosides.

Here, we describe detailed synthetic protocols for preparation of 6-amino/thio-2-triazolylpurine ribonucleosides. First, 9-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)-2,6-diazido-9H-purine, to be used as a key starting material, is synthesized in an SN Ar reaction with NaN3 starting from commercially available 9-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)-2,6-dichloro-9H-purine. Next, 2,6-bis-triazolylpurine ribonucleoside is obtained in a CuAAC reaction between diazidopurine derivative and phenyl acetylene, and used in SN Ar reactions with N- and S-nucleophiles. In these reactions, the triazolyl ring at the purine C6 position acts as a good leaving group. Cleavage of acetyl protecting groups from the ribosyl moiety is achieved in presence of piperidine. In the SN Ar reaction with amino acid derivatives, the acetyl groups remain intact. Moreover, 9-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)-2,6-diazido-9H-purine is selectively reduced at the C6 position using a CuSO4 ·5H2 O/sodium ascorbate system. This provides a straightforward approach for synthesis of 9-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)-6-amino-2-azido-9H-purine. © 2021 Wiley Periodicals LLC Basic Protocol 1: Synthesis of 6-amino-2-triazolylpurine ribonucleosides Basic Protocol 2: Synthesis of 6-thio-2-triazolylpurine ribonucleosides Basic Protocol 3: Synthesis of 6-amino-2-azidopurine ribonucleoside.

Identification of the Formycin A Biosynthetic Gene Cluster from Streptomyces kaniharaensis Illustrates the Interplay between Biological Pyrazolopyrimidine Formation and de Novo Purine Biosynthesis.

Formycin A is a potent purine nucleoside antibiotic with a C-glycosidic linkage between the ribosyl moiety and the pyrazolopyrimidine base. Herein, a cosmid is identified from the Streptomyces kaniharaensis genome library that contains the for gene cluster responsible for the biosynthesis of formycin. Subsequent gene deletion experiments and in vitro characterization of the forBCH gene products established their catalytic functions in formycin biosynthesis. Results also demonstrated that PurH from de novo purine biosynthesis plays a key role in pyrazolopyrimidine formation during biosynthesis of formycin A. The participation of PurH in both pathways represents a good example of how primary and secondary metabolism are interlinked.

Structural and Energetic Effects of O2'-Ribose Methylation of Protonated Purine Nucleosides.

The chemical difference between DNA and RNA nucleosides is their 2'-hydrogen versus 2'-hydroxyl substituents. Modification of the ribosyl moiety at the 2'-position and 2'-O-methylation in particular, is common among natural post-transcriptional modifications of RNA. 2'-Modification may alter the electronic properties and hydrogen-bonding characteristics of the nucleoside and thus may lead to enhanced stabilization or malfunction. The structures and relative glycosidic bond stabilities of the protonated forms of the 2'-O-methylated purine nucleosides, 2'-O-methyladenosine (Adom) and 2'-O-methylguanosine (Guom), were examined using two complementary tandem mass spectrometry approaches, infrared multiple photon dissociation action spectroscopy and energy-resolved collision-induced dissociation. Theoretical calculations were also performed to predict the structures and relative stabilities of stable low-energy conformations of the protonated forms of the 2'-O-methylated purine nucleosides and their infrared spectra in the gas phase. Low-energy conformations highly parallel to those found for the protonated forms of the canonical DNA and RNA purine nucleosides are also found for the protonated 2'-O-methylated purine nucleosides. Importantly, the preferred site of protonation, nucleobase orientation, and sugar puckering are preserved among the DNA, RNA, and 2'-O-methylated variants of the protonated purine nucleosides. The 2'-substituent does however influence hydrogen-bond stabilization as the 2'-O-methyl and 2'-hydroxyl substituents enable a hydrogen-bonding interaction between the 2'- and 3'-substituents, whereas a 2'-hydrogen atom does not. Further, 2'-O-methylation reduces the number of stable low-energy hydrogen-bonded conformations possible and importantly inverts the preferred polarity of this interaction versus that of the RNA analogues. Trends in the CID50% values extracted from survival yield analyses of the 2'-O-methylated and canonical DNA and RNA forms of the protonated purine nucleosides are employed to elucidate their relative glycosidic bond stabilities. The glycosidic bond stability of Adom is found to exceed that of its DNA and RNA analogues. The glycosidic bond stability of Guom is also found to exceed that of its DNA analogue; however, this modification weakens this bond relative to its RNA counterpart. The glycosidic bond stability of the protonated purine nucleosides appears to be correlated with the hydrogen-bond stabilization of the sugar moiety.

Catalytic strategy and inhibition of the prokaryotic specific GTP cyclohydrolase IB

GTP cyclohydrolase I catalyzes the first step in folic acid biosynthesis in bacteria and plants, biopterin biosynthesis in mammals, and the biosynthesis of 7‐deazaguanosine modified tRNA nucleosides in bacteria and archaea. The type IB GTP cyclohydrolase (GCYH‐IB) is a prokaryotic‐specific enzyme found in several pathogens. GCYH‐IB is structurally distinct from the canonical type IA GTP cyclohydrolase involved in biopterin biosynthesis in humans and animals, and thus is of interest as a potential antibacterial drug target. We report kinetic and inhibition data of Neisseria gonorrhoeae (Ng) GCYH‐IB, and two high‐resolution crystal structures of the enzyme; one in complex with the reaction intermediate analog and a competitive inhibitor 8‐oxo‐GTP, and one with a TRIS molecule bound in the active site which mimics another reaction intermediate. Comparison of these GCYH‐IB crystal structures with the type IA enzyme bound to 8‐oxo‐GTP, reveals an inverted binding mode of the inhibitor's ribosyl moiety. Taken together with site‐directed mutagenesis data, suggests that the two enzymes utilize different strategies for catalysis. Additionally, the inhibitor interacts with a conserved active site residue Cys149 which is S‐nitrosylated in the structures. This is the first structural characterization of a biologically S‐nitrosylated bacterial protein. Mutagenesis and biochemical analyses demonstrate that Cys149 is essential for the cyclohydrolase reaction, and S‐nitrosylation maintains enzyme activity, suggesting a potential role of the S‐nitrosothiol in catalysis. These structures and information were used to perform structure based drug design using a combination of in silico screening and de novo design methods to generate molecules that inhibit NgGCYH‐IB selectively over human GCYH‐IA. A few compounds named G1, G2 and G1‐triphosphate have been generated and currently being tested for inhibition.Support or Funding InformationThis project was supported by NSF grant CHE‐1309323 to D. Iwata‐Reuyl and M.A. Swairjo,NIH grant GM110588 to M.A. Swairjo and D. Iwata‐Reuyl, NIH grant GM70641 to D. Iwata‐Reuyl, andan intramural grant from Western University of Health Sciences to M.A. Swairjo.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanism and catalytic strategy of the prokaryotic-specific GTP cyclohydrolase-IB.

Guanosine 5'-triphosphate (GTP) cyclohydrolase-I (GCYH-I) catalyzes the first step in folic acid biosynthesis in bacteria and plants, biopterin biosynthesis in mammals, and the biosynthesis of 7-deazaguanosine-modified tRNA nucleosides in bacteria and archaea. The type IB GCYH (GCYH-IB) is a prokaryotic-specific enzyme found in many pathogens. GCYH-IB is structurally distinct from the canonical type IA GCYH involved in biopterin biosynthesis in humans and animals, and thus is of interest as a potential antibacterial drug target. We report kinetic and inhibition data of Neisseria gonorrhoeae GCYH-IB and two high-resolution crystal structures of the enzyme; one in complex with the reaction intermediate analog and competitive inhibitor 8-oxoguanosine 5'-triphosphate (8-oxo-GTP), and one with a tris(hydroxymethyl)aminomethane molecule bound in the active site and mimicking another reaction intermediate. Comparison with the type IA enzyme bound to 8-oxo-GTP (guanosine 5'-triphosphate) reveals an inverted mode of binding of the inhibitor ribosyl moiety and, together with site-directed mutagenesis data, shows that the two enzymes utilize different strategies for catalysis. Notably, the inhibitor interacts with a conserved active-site Cys149, and this residue is S-nitrosylated in the structures. This is the first structural characterization of a biologically S-nitrosylated bacterial protein. Mutagenesis and biochemical analyses demonstrate that Cys149 is essential for the cyclohydrolase reaction, and S-nitrosylation maintains enzyme activity, suggesting a potential role of the S-nitrosothiol in catalysis.

Alteration in substrate specificity of horse liver alcohol dehydrogenase by an acyclic nicotinamide analog of NAD(+).

A new, acyclic NAD-analog, acycloNAD(+) has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of β-NAD(+) with a redox potential of -324mV and a 341nm λmax for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD(+) by primary alcohols. With HLADH 1-butanol has the highest Vmax at 49% that of β-NAD(+). The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon-hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with β-NAD(+). In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD(+) by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD(+) has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with α-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases.

The crystal structure of type III effector protein XopQ from Xanthomonas oryzae complexed with adenosine diphosphate ribose.

Effector proteins are virulence factors that promote pathogenesis by interfering with various cellular events and are delivered directly into host cells by the secretion systems of many Gram-negative bacteria. Type III effector protein XOO4466 from the plant pathogen Xanthomonas oryzae pv. oryzae (XopQ(Xoo)) and XopQ homologs from other phytopathogens have been predicted to be nucleoside hydrolases based on their sequence similarities. However, despite such similarities, recent structural and functional studies have revealed that XopQ(Xoo) does not exhibit the expected activity of a nucleoside hydrolase. On the basis of the conservation of a Ca(2+) coordination shell of a ribose-binding site and the spacious active site in XopQ(Xoo), we hypothesized that a novel compound containing a ribosyl moiety could serve as a substrate for XopQ(Xoo). Here, we report the crystal structure of XopQ(Xoo) in complex with adenosine diphosphate ribose (ADPR), which is involved in regulating cytoplasmic Ca(2+) concentrations in eukaryotic cells. ADPR is bound to the active site of XopQ(Xoo) with its ribosyl end tethered to the Ca(2+) coordination shell. The binding of ADPR is further stabilized by interactions mediated by hydrophobic residues that undergo ligand-induced conformational changes. These data showed that XopQ(Xoo) is capable of binding a novel chemical bearing a ribosyl moiety, thereby providing the first step toward understanding the functional role of XopQ(Xoo).

Structure/Activity Relationships of (M)ANT- and TNP-Nucleotides for Inhibition of Rat Soluble Guanylyl Cyclase α1β1

Soluble guanylyl cyclase (sGC) plays an important role in cardiovascular function and catalyzes formation of cGMP. sGC is activated by nitric oxide and allosteric stimulators and activators. However, despite its therapeutic relevance, the regulatory mechanisms of sGC are still incompletely understood. A major reason for this situation is that no crystal structures of active sGC have been resolved so far. An important step toward this goal is the identification of high-affinity ligands that stabilize an sGC conformation resembling the active, "fully closed" state. Therefore, we examined inhibition of rat sGCα1β1 by 38 purine- and pyrimidine-nucleotides with 2,4,6,-trinitrophenyl and (N-methyl)anthraniloyl substitutions at the ribosyl moiety and compared the data with that for the structurally related membranous adenylyl cyclases (mACs) 1, 2, 5 and the purified mAC catalytic subunits VC1:IIC2. TNP-GTP [2',3'-O-(2,4,6-trinitrophenyl)-GTP] was the most potent sGCα1β1 inhibitor (Ki, 10.7 nM), followed by 2'-MANT-3'-dATP [2'-O-(N-methylanthraniloyl)-3'-deoxy-ATP] (Ki, 16.7 nM). Docking studies on an sGCαcat/sGCβcat model derived from the inactive heterodimeric crystal structure of the catalytic domains point to similar interactions of (M)ANT- and TNP-nucleotides with sGCα1β1 and mAC VC1:IIC2. Reasonable binding modes of 2'-MANT-3'-dATP and bis-(M)ANT-nucleotides at sGC α1β1 require a 3'-endo ribosyl conformation (versus 3'-exo in 3'-MANT-2'-dATP). Overall, inhibitory potencies of nucleotides at sGCα1β1 versus mACs 1, 2, 5 correlated poorly. Collectively, we identified highly potent sGCα1β1 inhibitors that may be useful for future crystallographic and fluorescence spectroscopy studies. Moreover, it may become possible to develop mAC inhibitors with selectivity relative to sGC.

An Active Site Water Broadens Substrate Specificity in S-Ribosylhomocysteinase (LuxS): A Docking, MD, and QM/MM Study

Type-2 quorum sensing (QS-2) is a cell-cell signaling process known to be used by a number of pathogenic bacteria and to play important roles in their population growth and virulence. S-ribosyl homocysteinase (LuxS) is a key enzyme in the formation of the signaling molecule of QS-2, autoinducer II (AI-2). In this study, substrate (S-ribosylhomocysteine: SRH) binding and possible initial reaction steps of its catalytic mechanism leading to formation of a putative 2-keto-SRH intermediate have been examined. Specifically, docking and MD simulations were used to gain insights into the structure of the active-site-bound substrate complex. An ONIOM QM/MM hybrid method was then used to elucidate the mechanism of the first stage of the enzyme catalytic process. It is shown that the substrate may bind within the active site when its ribosyl moiety is in the α- (α-SRH) or β-furanose (β-SRH) configuration or as a linear aldose (linear-SRH). The α-SRH complex is preferred, lying 47.5 kJ mol(-1) lower in energy than the next lowest energy initial complex β-SRH. However, the MD and QM/MM calculations indicate that an active site water stably locates within the active site and that it can facilitate ring-opening of either α-SRH or β-furanose, leading to formation of a common active-site-bound 2-keto-SRH intermediate, without the need to pass through a linear aldose SRH configuration. Hence, regardless of the ribose's configuration within the bound SRH substrate, LuxS is able to catalyze the conversion of SRH to a common 2-keto-SRH intermediate.

Transition-State Analysis of Trypanosoma cruzi Uridine Phosphorylase-Catalyzed Arsenolysis of Uridine

Uridine phosphorylase catalyzes the reversible phosphorolysis of uridine and 2'-deoxyuridine to generate uracil and (2-deoxy)ribose 1-phosphate, an important step in the pyrimidine salvage pathway. The coding sequence annotated as a putative nucleoside phosphorylase in the Trypanosoma cruzi genome was overexpressed in Escherichia coli , purified to homogeneity, and shown to be a homodimeric uridine phosphorylase, with similar specificity for uridine and 2'-deoxyuridine and undetectable activity toward thymidine and purine nucleosides. Competitive kinetic isotope effects (KIEs) were measured and corrected for a forward commitment factor using arsenate as the nucleophile. The intrinsic KIEs are: 1'-(14)C = 1.103, 1,3-(15)N(2) = 1.034, 3-(15)N = 1.004, 1-(15)N = 1.030, 1'-(3)H = 1.132, 2'-(2)H = 1.086, and 5'-(3)H(2) = 1.041 for this reaction. Density functional theory was employed to quantitatively interpret the KIEs in terms of transition-state structure and geometry. Matching of experimental KIEs to proposed transition-state structures suggests an almost synchronous, S(N)2-like transition-state model, in which the ribosyl moiety possesses significant bond order to both nucleophile and leaving groups. Natural bond orbital analysis allowed a comparison of the charge distribution pattern between the ground-state and the transition-state models.

Characterization of LipL as a Non-heme, Fe(II)-dependent α-Ketoglutarate:UMP Dioxygenase That Generates Uridine-5′-aldehyde during A-90289 Biosynthesis

Fe(II)- and α-ketoglutarate (α-KG)-dependent dioxygenases are a large and diverse superfamily of mononuclear, non-heme enzymes that perform a variety of oxidative transformations typically coupling oxidative decarboxylation of α-KG with hydroxylation of a prime substrate. The biosynthetic gene clusters for several nucleoside antibiotics that contain a modified uridine component, including the lipopeptidyl nucleoside A-90289 from Streptomyces sp. SANK 60405, have recently been reported, revealing a shared open reading frame with sequence similarity to proteins annotated as α-KG:taurine dioxygenases (TauD), a well characterized member of this dioxygenase superfamily. We now provide in vitro data to support the functional assignment of LipL, the putative TauD enzyme from the A-90289 gene cluster, as a non-heme, Fe(II)-dependent α-KG:UMP dioxygenase that produces uridine-5'-aldehyde to initiate the biosynthesis of the modified uridine component of A-90289. The activity of LipL is shown to be dependent on Fe(II), α-KG, and O(2), stimulated by ascorbic acid, and inhibited by several divalent metals. In the absence of the prime substrate UMP, LipL is able to catalyze oxidative decarboxylation of α-KG, although at a significantly reduced rate. The steady-state kinetic parameters using optimized conditions were determined to be K(m)(α-KG) = 7.5 μM, K(m)(UMP) = 14 μM, and k(cat) ≈ 80 min(-1). The discovery of this new activity not only sets the stage to explore the mechanism of LipL and related dioxygenases further but also has critical implications for delineating the biosynthetic pathway of several related nucleoside antibiotics.

The Crystal Structure and Activity of a Putative Trypanosomal Nucleoside Phosphorylase Reveal It to be a Homodimeric Uridine Phosphorylase

Purine nucleoside phosphorylases (PNPs) and uridine phosphorylases (UPs) are closely related enzymes involved in purine and pyrimidine salvage, respectively, which catalyze the removal of the ribosyl moiety from nucleosides so that the nucleotide base may be recycled. Parasitic protozoa generally are incapable of de novo purine biosynthesis; hence, the purine salvage pathway is of potential therapeutic interest. Information about pyrimidine biosynthesis in these organisms is much more limited. Though all seem to carry at least a subset of enzymes from each pathway, the dependency on de novo pyrimidine synthesis versus salvage varies from organism to organism and even from one growth stage to another. We have structurally and biochemically characterized a putative nucleoside phosphorylase (NP) from the pathogenic protozoan Trypanosoma brucei and find that it is a homodimeric UP. This is the first characterization of a UP from a trypanosomal source despite this activity being observed decades ago. Although this gene was broadly annotated as a putative NP, it was widely inferred to be a purine nucleoside phosphorylase. Our characterization of this trypanosomal enzyme shows that it is possible to distinguish between PNP and UP activity at the sequence level based on the absence or presence of a characteristic UP-specificity insert. We suggest that this recognizable feature may aid in proper annotation of the substrate specificity of enzymes in the NP family.

Photocycloaddition of the T1 excited state of thioinosine to uridine and adenosine

Novel photoadducts were obtained by irradiation of thioinosine (6-thiopurine riboside, TI) in deaerated aqueous solution without and in the presence of uridine and adenosine. Excitation (lambda > 300 nm) of TI to its excited S2 state yields a single bimolecular photoproduct. It is a purine-pyrimidine diriboside in which the purine ring is attached to the amide nitrogen of 6-amino-4-thioxo-5-formamidopyrimidine. When TI was irradiated in the presence of an excess of adenosine, two photoproducts were isolated: diribosides of N-(4,6-diaminopirymidin-5-yl)-N-formyl-6-aminopurine and N-(4-amino-6-formylamino-pyrimidin-5-yl)-6-aminopurine, both containing a purine and a formylaminopyrimidine (Fapy) fragment. The photoreaction of TI with uridine gave two regioisomeric photoproducts identified as diribosides containing either 5- or 6-(purin-6-yl)uracil as aglycones. A multistep mechanism leading to the stable photoproducts is proposed. In the first step of the mechanism, the C=S group of the excited TI undergoes a [2 + 2] cycloaddition regioselectively to the N(7)=C(8) bond of the purine ring or adds in a non-regioselective manner to the C(5)=C(6) bond of uracil. The unstable photoproducts thus formed undergo a series of dark reactions at room temperature. The photocycloaddition reactions originate from the excited T1 state of TI. This conclusion is supported by a combination of evidence from reaction quenching studies using both steady-state quantum yield determinations and kinetics results from nanosecond laser flash photolysis. The T1 state of TI is quenched by other TI molecules in their S0 state (self-quenching) and also by uridine and adenosine, all with large rate constants (0.8-5) x 10(9) M(-1) s(-1). The quantum yields of the reactions are in general very low (phi(R) < or = 8 x 10(-3)). The sources of the inefficiency in the photocycloaddition of TI to uridine and adenosine are discussed. The photoproducts containing the Fapy residue undergo deformylation and isomerization of the ribosyl moiety (anomerization, furanose/pyranose transformation) upon heating in aqueous solution. Products of the transformations were identified.

Formation of a Sandwich-Structure Assisted, Relatively Long-Lived Sulfur-Centered Three-Electron Bonded Radical Anion in the Reduction of a Bis(1-substituted-uracilyl) Disulfide in Aqueous Solution

The one-electron reduction of bis[1-(2',3',5'-tri-O-acetylribosyl)uracil-4-yl] disulfide, initiated by hydrated electrons in a radiation chemical study, has been shown to yield 1-(2',3',5'-tri-O-acetylribosyl)-4-thiouracil as a stable molecular product. The reduction reaction leads, in the first instance, to a transient, albeit remarkably stable disulfide radical anion. This is characterized by a 2-center-3-electron bond with two bonding sigma-electrons and an antibonding sigma*-electron in the sulfur-sulfur bridge, (-S therefore S-)(-). It receives its stability from a sandwich-structure with the two uracilyl moieties facing each other (possibly further assisted by the 2',3',5'-tri-O-acetylribosyl substituents). A considerable lengthening of the original disulfide bridge from 2.02 to 2.73 A in the radical anion seems to facilitate the interaction of the heterocycles and leads to a gain in stabilization energy of 24 and 33 kcal/mol (100 and 140 kJ/mol) as evaluated by UMP2/cc-pVTZ and UMP2/cc-pVDZ calculations, respectively. The (-S therefore S-)(-) bonded radical anion shows a broad optical absorption band with lambdamax=450 nm, epsilonmax=6000 M(-1) cm(-1), and a half-width of 1.0 eV. It exists in equilibrium with the conjugated 1-(2',3',5'-tri- O-acetylribosyl)uracil-4-yl thiyl radical -S(*), and the corresponding thiolate, -S(-). The rate determining step for the disappearance of the disulfide radical anion appears to be protonation of both the radical anion and the free thiolate by reaction with H(+)aq. Absolute rate constants have been measured for these protonation processes, for the formation of the stable thiouridine product, and for the electron transfer from the disulfide radical anion to molecular oxygen. With the (-S therefore S-)(-) <--> -S(*) + -S(-) equilibrium lying very much on the left-hand side, the reduced disulfide system exhibits predominantly reducing properties whereas any oxidizing property of the conjugated thiyl radical has only little if any chance to materialize. Besides attaching directly to the disulfide bridge, the hydrated electrons react also, with about equal efficiency, with the uracil moiety of the investigated compound. This leads to a structurally totally different and electronically distinguishable species than that with the reduced disulfide bridge. In particular, there is no face-to-face interaction between the two heterocyclic moieties and no increased electron density in the S-S bond. The C-centered radicals resulting from the reduction of the uracil and possibly also generated from the ribosyl moieties initiate further cleavage of the S-S bond and thus contribute to the formation of thiouridine. The overall yield of the latter, as determined from steady-state gamma-radiolysis, indicates a small chain process (G=1.54 micromol/J). Possible mechanisms are discussed.