Nucleotide Acceptor Research Articles

Evidences for reaction mechanism of 9DB1 DNA catalyst

The first reported reaction mechanism of a DNAzyme, i.e. 9DB1, by using molecular dynamics (MD) simulations includes some ambiguities. We try to overcome some of these ambiguous aspects such as the role of mono and divalent metal ions and observed metal rescue effects by surveying the role of functional groups of original 9DB1 and a variety of its rate conserving and rate decreasing mutations via MD simulations. Conformational differences of these two distinct groups are responsible for their opposite rate trends. Blocking of the OH3’ of acceptor nucleotide from effective attack by its hydrogen bond to O4’ of donor nucleotide is observed in rate decreasing mutations. Our simulations manifest the role of Na+ and Mg2+ ions in bringing close to each other the ligated atoms. These findings along with observed conformational changes explain carefully the reported metal rescue effects for some phosphate groups.

Structural Basis for the Stereochemical Control of Amine Installation in Nucleotide Sugar Aminotransferases.

Sugar aminotransferases (SATs) are an important class of tailoring enzymes that catalyze the 5'-pyridoxal phosphate (PLP)-dependent stereo- and regiospecific installation of an amino group from an amino acid donor (typically L-Glu or L-Gln) to a corresponding ketosugar nucleotide acceptor. Herein we report the strategic structural study of two homologous C4 SATs (Micromonospora echinospora CalS13 and Escherichia coli WecE) that utilize identical substrates but differ in their stereochemistry of aminotransfer. This study reveals for the first time a new mode of SAT sugar nucleotide binding and, in conjunction with previously reported SAT structural studies, provides the basis from which to propose a universal model for SAT stereo- and regiochemical control of amine installation. Specifically, the universal model put forth highlights catalytic divergence to derive solely from distinctions within nucleotide sugar orientation upon binding within a relatively fixed SAT active site where the available ligand bound structures of the three out of four representative C3 and C4 SAT examples provide a basis for the overall model. Importantly, this study presents a new predictive model to support SAT functional annotation, biochemical study and rational engineering.

Structural Basis for the Catalytic Mechanism of DncV, Bacterial Homolog of Cyclic GMP-AMP Synthase

Cyclic dinucleotides (CDNs) play key roles as second messengers and signaling molecules in bacteria andmetazoans. The newly identified dinucleotide cyclase in Vibrio cholerae (DncV) produces three different CDNs containing two 3'-5' phosphodiester bonds, and its predominant product is cyclic GMP-AMP, whereas mammalian cyclic GMP-AMP synthase (cGAS) produces only cyclic GMP-AMP containing mixed 2'-5' phosphodiester bonds. We report the crystal structures of V.cholerae and Escherichia coli DncV in complex with various nucleotides in the pre-reaction states. The high-resolution structures revealed that DncV preferably recognizes ATP and GTP as acceptor and donor nucleotides, respectively, in the first nucleotidyl transfer reaction. Considering the recently reported intermediate structures, our pre-reaction state structures providethe precise mechanism of 3'-5' linked cyclic AMP-GMP production in bacteria. A comparison with cGAS in the pre-reaction states suggests that the orientation of the acceptor nucleotide primarily determines the distinct linkage specificities between DncV and cGAS.

Solution structure of the porcine sapovirus VPg core reveals a stable three-helical bundle with a conserved surface patch

Viral protein genome-linked (VPg) proteins play a critical role in the life cycle of vertebrate and plant positive-sense RNA viruses by acting as a protein primer for genome replication and as a protein cap for translation initiation. Here we report the solution structure of the porcine sapovirus VPg core (VPgC) determined by multi-dimensional NMR spectroscopy. The structure of VPgC is composed of three α-helices stabilized by several conserved hydrophobic residues that form a helical bundle core similar to that of feline calicivirus VPg. The putative nucleotide acceptor Tyr956 within the first helix of the core is completely exposed to solvent accessible surface to facilitate nucleotidylation by viral RNA polymerase. Comparison of VPg structures suggests that the surface for nucleotidylation site is highly conserved among the Caliciviridae family, whereas the backbone core structures are different. These structural features suggest that caliciviruses share common mechanisms of VPg-dependent viral replication and translation.

Structural basis for the replication of genomic RNA of porcine enteric sapovirus by VPg (LB243)

Structural basis for the replication of genomic RNA of porcine enteric sapovirus by VPg Hye Jung Min, Hyo‐Jeong Hwang, Jeong‐Sun Kim, and Chul Won Lee Department of Chemistry, Chonnam National University, Gwangju 500‐757, Korea. Viral protein genome‐linked (VPg) plays a critical role in the life cycle of vertebrate and plant positive‐sense (+) RNA viruses by acting as a protein primer for RNA synthesis. The replication of the viruses is primed by the VPg protein by attaching to the 5’‐end of the viral RNA genome. Here we show the solution structure of VPg of porcine enteric sapovirus (PSV) determined by multi‐dimensional NMR spectroscopy. The structure of VPg comprises three helices (denoted α1, α2, and α3) which were tightly packed together through hydrophobic interactions formed by several hydrophobic residues to form a three‐helix bundle structure. The orientation and length of three helices of PSV VPg are similar to those of feline calicivirus (FCV) VPg with similar core structure formed by conserved hydrophobic residues. The tyrosine residue (Tyr956) within the conserved motif for nucleotide acceptor is exposed to the solvent surface to facilitate nucleotidylation by viral RNA polymerases. These results may provide the molecular basis for the replication of positive‐RNA viruses.

A Solanum tuberosum inositol phosphate kinase (StITPK1) displaying inositol phosphate–inositol phosphate and inositol phosphate–ADP phosphotransferase activities

We describe a multifunctional inositol polyphosphate kinase/phosphotransferase from Solanum tuberosum, StITPKα (GenBank accession number: EF362784), hereafter called StITPK1. StITPK1 displays inositol 3,4,5,6-tetrakisphosphate 1-kinase activity: K m = 27 μM, and V max = 19 nmol min −1 mg −1. The enzyme displays inositol 1,3,4,5,6-pentakisphosphate 1-phosphatase activity in the absence of a nucleotide acceptor and inositol 1,3,4,5,6-pentakisphosphate–ADP phosphotransferase activity in the presence of physiological concentrations of ADP. Additionally, StITPK1 shows inositol phosphate-inositol phosphate phosphotransferase activity. Homology modelling provides a structural rationale of the catalytic abilities of StITPK1. Inter-substrate transfer of phosphate groups between inositol phosphates is an evolutionarily conserved function of enzymes of this class.

Structural Rearrangements in the Active Site of Smooth-Muscle Myosin

Structural rearrangements of the myosin upper-50 kD subdomain are thought to play a key role in coordinating actin binding with nucleotide hydrolysis during the myosin ATPase cycle. Such rearrangements could open and close the active site in opposition to the actin-binding cleft, helping explain the opposing affinities of myosin for actin and nucleotide. To directly examine conformational changes across the active site during the ATPase cycle we have genetically engineered a mutant of chicken smooth-muscle myosin, F344W motor domain essential light chain, which contains a single tryptophan (344W) located on a short loop between two alpha helixes that traverse the upper-50 kD subdomain in front of the active site. Fluorescence resonance energy transfer was examined between the 344W donor probe and 2′(3′)-O-( N-methylanthraniloyl) (mant)-nucleotide acceptor probes in the active site of this construct. The observed fluorescence resonance energy transfer efficiencies were 6.4% in the presence of mant ADP and 23.8% in the presence of mant ATP, corresponding to distances of 33.4 Å and 24.9 Å, respectively. Our results are consistent with structural rearrangements in which there is an 8.5-Å closure between the 344W residue and the mant moiety during the transition from the strongly (ADP) to weakly (ATP) actin-bound states of the myosin ATPase cycle.

Structure of Escherichia coli UMP Kinase Differs from That ofOther Nucleoside Monophosphate Kinases and Sheds New Light on EnzymeRegulation

Structure of Escherichia coli UMP Kinase Differs from That ofOther Nucleoside Monophosphate Kinases and Sheds New Light on EnzymeRegulation

PH opposite effects on synthesis of dinucleoside polyphosphates and on oxidation reactions catalyzed by firefly luciferase

Previous results have shown that an oxidizing product of firefly luciferin, dehydroluciferyl-adenylate, is the main intermediate in the process of synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase (EC 1.13.12.7). However, we have found that the pH effects on the luciferase oxidizing processes and on the synthesis of dinucleoside polyphosphate are opposite: acidic assay media enhance the synthesis of dinucleoside polyphosphate and inhibit the oxidizing processes. The reason for this apparent contradiction lies on the activation effect of low pH on the adenylate transfer reaction from dehydroluciferyl-adenylate to the acceptor nucleotide.

Kinetoplastid RNA editing ligases: complex association, characterization, and substrate requirements.

RNA editing processes kinetoplastid mitochondrial transcripts post-transcriptionally by inserting and deleting uridylates (Us) to produce functional mRNAs. The activities of the RNA ligases in the multienzyme complex (the editosome) that catalyzes editing and of the recombinant proteins were characterized and found to be similar. Ligation of two RNA fragments was enhanced when bridged by a complementary RNA or DNA, which left no gaps or overhangs. An acceptor nucleotide preference of G>U>C>A was observed in the absence of exogenous ATP but U was preferred upon addition of ATP and ligase activity was increased. The substrate specificity and catalytic characteristics indicate that RNA ligase activity contributes to the accuracy of RNA editing.

Nucleotide affinity for a stable phosphorylated intermediate of nucleoside diphosphate kinase.

Nucleoside diphosphate (NDP) kinase is transiently phosphorylated on a histidine of the active site during the catalytic cycle. In the presence of a nucleotide acceptor, the phosphohistidine bond is unstable and the phosphate is transferred to the acceptor in less than 1 msec. We describe the synthesis of an analog of the phosphoenzyme intermediate with an inactive mutant of NDP kinase in which the catalytic histidine is replaced by a cysteine. In two sequential disulfide exchange reactions, a thiophosphate group reacts with the thiol function of the cysteine that had previously reacted with dithionitrobenzoate (DTNB). The thiophosphoenzyme presents a 400,000-fold increased stability in the presence of NDPs compared with the phosphoenzyme. The binding of NDP is studied at the steady state and presteady state. Data were analyzed according to a bimolecular association model. For the first time, the true equilibrium dissociation constants of NDP for the analog of the phosphoenzyme are determined in the absence of phosphotransfer, allowing a better understanding of the catalytic mechanism of the enzyme.

Differential removal of thymidine nucleotide analogues from blocked DNA chains by human immunodeficiency virus reverse transcriptase in the presence of physiological concentrations of 2'-deoxynucleoside triphosphates.

Removal of 2',3'-didehydro-3'-deoxythymidine-5'-monophosphate (d4TMP) from a blocked DNA chain can occur through transfer of the chain-terminating residue to a nucleotide acceptor by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). ATP-dependent removal of either d4TMP or 3'-azido-3'-deoxythymidine-5'-monophosphate (AZTMP) is increased in AZT resistant HIV-1 RT (containing D67N/K70R/T215F/K219Q mutations). Removal of d4TMP is strongly inhibited by the next complementary deoxynucleoside triphosphate (50% inhibitory concentration [IC(50)] of approximately 0.5 microM), whereas removal of AZTMP is much less sensitive to this inhibition (IC(50) of >100 microM). This could explain the lack of cross-resistance by AZT-resistant HIV-1 to d4T in phenotypic drug susceptibility assays.

Phosphorylation or oxydation level of the phosphate acceptor nucleotide modifies crystal structure ofE. coliCMP kinase: correlation with kinetics and site-directed mutagenesis

Phosphorylation or oxydation level of the phosphate acceptor nucleotide modifies crystal structure of<i>E. coli</i>CMP kinase: correlation with kinetics and site-directed mutagenesis

Inefficient Spliceosome Assembly and Abnormal Branch Site Selection in Splicing of an HIV-1 Transcript in Vitro

Continuous replication of human immunodeficiency virus type I (HIV-1) requires balanced expression of spliced and nonspliced mRNAs in the cytoplasm. This process is regulated post-transcriptionally by the viral-encoded Rev protein. An important prerequisite for Rev responsiveness is the presence of weak splice sites in the viral mRNA. We have investigated the splicing of the second intron of the HIV-1 Tat/Rev transcript in vitro and show that the 3'-splice site region is responsible for the inefficient splicing of the HIV-1 transcript. In contrast, the HIV-1 5'-splice site is highly functional in combination with a heterologous 3'-splice site. Incubation of the HIV-1 transcript in nuclear extract leads to a rapid accumulation of 50 S nonproductive pre-spliceosome complexes. These complexes contain mainly U1 and U2 small nuclear ribonucleoproteins and are formed independently of the presence of the downstream 3'-splice site. The HIV-1 transcripts, which do proceed through the first splicing step, utilize primarily a uridine as the branch acceptor nucleotide. Sequence comparison with other HIV-1 introns suggests that nucleotides other than adenosines are commonly used as branch points in these viruses.

Two different sequence elements within exon 4 are necessary for calcitonin-specific splicing of the human calcitonin/calcitonin gene-related peptide I pre-mRNA.

The calcitonin (CT)/calcitonin gene-related peptide I (CGRP-I) gene (CALC-I gene) is subject to alternative tissue-specific processing of its primary transcript. CT mRNA is the predominant mRNA produced in thyroid C cells, whereas CT gene-related peptide I mRNA is the main product in neurons of the central and peripheral nervous systems. The CT-specific exon 4 is surrounded by weak processing sites. In this study we have investigated whether exon 4 sequences are involved in the tissue-specific selection of the exon 4 splice acceptor site. The results indicate that two separate elements, termed A and B, in the 5' part of exon 4 are required for production of CT-specific RNA. These sequences are located between nucleotides 67 and 88 (A) and nucleotides 117 and 146 (B) relative to the 5' end of exon 4. Variation of the distance between these sequence elements and the 3' splice site of exon 4 does not change the processing choice. These sequence elements are functionally equivalent. CT-specific splicing requires the presence of both sequence A and B or duplicates of either sequence element in exon 4. The effect of these sequences on the RNA processing choice is overruled by mutation of the CT-specific uridine branch acceptor nucleotide into a commonly preferred adenosine residue.

Two different sequence elements within exon 4 are necessary for calcitonin-specific splicing of the human calcitonin/calcitonin gene-related peptide I pre-mRNA.

The calcitonin (CT)/calcitonin gene-related peptide I (CGRP-I) gene (CALC-I gene) is subject to alternative tissue-specific processing of its primary transcript. CT mRNA is the predominant mRNA produced in thyroid C cells, whereas CT gene-related peptide I mRNA is the main product in neurons of the central and peripheral nervous systems. The CT-specific exon 4 is surrounded by weak processing sites. In this study we have investigated whether exon 4 sequences are involved in the tissue-specific selection of the exon 4 splice acceptor site. The results indicate that two separate elements, termed A and B, in the 5' part of exon 4 are required for production of CT-specific RNA. These sequences are located between nucleotides 67 and 88 (A) and nucleotides 117 and 146 (B) relative to the 5' end of exon 4. Variation of the distance between these sequence elements and the 3' splice site of exon 4 does not change the processing choice. These sequence elements are functionally equivalent. CT-specific splicing requires the presence of both sequence A and B or duplicates of either sequence element in exon 4. The effect of these sequences on the RNA processing choice is overruled by mutation of the CT-specific uridine branch acceptor nucleotide into a commonly preferred adenosine residue.

Unusual branch point selection in processing of human growth hormone pre-mRNA.

Intron A of the human growth hormone gene does not contain an A residue within 56 nucleotides preceding the 3' splice site. The analysis of the excised intron lariat revealed a C residue 28 nucleotides upstream from the 3' splice site as the major branch acceptor nucleotide. Two additional minor branched nucleotides were identified as U residues at positions -22 and -36. An adenosine substitution at position -22 results in lariat formation solely to this nucleotide. Therefore, C and U residues can function efficiently as natural branch acceptors, but an A residue is preferred if available in the proper region. In addition, the data strongly reinforce the importance of the distance constraint for lariat formation. To explain selection of the branch acceptor nucleotide, potential base-pairing interactions of branch point sequences with the U2 RNA are discussed.

Unusual branch point selection in processing of human growth hormone pre-mRNA.

Intron A of the human growth hormone gene does not contain an A residue within 56 nucleotides preceding the 3' splice site. The analysis of the excised intron lariat revealed a C residue 28 nucleotides upstream from the 3' splice site as the major branch acceptor nucleotide. Two additional minor branched nucleotides were identified as U residues at positions -22 and -36. An adenosine substitution at position -22 results in lariat formation solely to this nucleotide. Therefore, C and U residues can function efficiently as natural branch acceptors, but an A residue is preferred if available in the proper region. In addition, the data strongly reinforce the importance of the distance constraint for lariat formation. To explain selection of the branch acceptor nucleotide, potential base-pairing interactions of branch point sequences with the U2 RNA are discussed.

A role for branchpoints in splicing in vivo.

The nucleotides immediately surrounding intron/exon junctions of genes transcribed by RNA polymerase B can be derived from 'consensus' sequences for donor and acceptor splice sites by only a few base changes. Studies in vivo have underlined the importance of these junction nucleotides for splicing. In higher eukaryotes, no evidence has been found for specific internal intron sequences involved in splicing. However, the recent discovery that, in vitro, introns are excised in a lariat form where the 5' end of the intron is joined via a 2'-5'-phosphodiester linkage to an A residue (branchpoint acceptor) close to the 3' end of the intron, suggests that internal intron sequences may nonetheless be important for splicing. Indeed, in yeast nuclear genes, the internal sequence 5'-TACTAAC-3' (or close homologue) is essential for splicing in vivo. A proposed consensus sequence for branchpoints in mammalian introns is 5'-CT(A/G)A(C/T)-3'. This sequence resembles the essential yeast internal sequence. Are branchpoints involved in the splicing of introns of higher eukaryotes in vivo? We show here that a branchpoint sequence from a human globin gene (5'-CTGACTCTCTCTG-3') greatly enhances the efficiency of splicing of a 'synthetic' intron in HeLa cells. A mutated branchpoint sequence, 5'-CTCCTCTCTCTG-3', in which the branchpoint acceptor nucleotide A has been deleted and the neighbouring purine G mutated to a C, does not exhibit this enhancing capability. We conclude that branchpoints have an important function in the splicing process in vivo.

Pig heart nucleosidediphosphate kinase. Phosphorylation and interaction with Cibacron blue 3GA.

The nucleosidediphosphate kinase phosphorylation reaction led to the incorporation of 0.95 +/- 0.1 phosphate groups per enzyme subunit. The equilibrium constant of the phosphorylation reaction was 0.26. The inhibition of the nucleosidediphosphate kinase activity by Cibacron blue 3GA was competitive with respect to ATP, the donor nucleotide (apparent Ki = 0.28 microM) and uncompetitive with respect to 8-bromoinosine 5'-diphosphate, the acceptor nucleotide (apparent Ki = 0.31 microM). By difference spectroscopy it was shown that each enzyme subunit bound one Cibacron blue 3GA molecule, whereas the phosphorylated enzyme had no affinity for the dye. ATP was an effective competitor, being able to displace the dye from its bound state. The complex behaviour noted was taken as evidence for cooperative interaction between the enzyme subunits. The data obtained using polarographic techniques agreed with these results.