The Experts below are selected from a list of 159 Experts worldwide ranked by ideXlab platform
Stewart Shuman - One of the best experts on this subject based on the ideXlab platform.
-
Structure-function analysis of Plasmodium RNA Triphosphatase and description of a triphosphate tunnel metalloenzyme superfamily that includes Cet1-like RNA Triphosphatases and CYTH proteins.
RNA (New York N.Y.), 2006Co-Authors: Chunling Gong, Paul Smith, Stewart ShumanAbstract:RNA Triphosphatase catalyzes the first step in mRNA capping. The RNA Triphosphatases of fungi and protozoa are structurally and mechanistically unrelated to the analogous mammalian enzyme, a situation that recommends RNA Triphosphatase as an anti-infective target. Fungal and protozoan RNA Triphosphatases belong to a family of metal-dependent phosphohydrolases exemplified by yeast Cet1. The Cet1 active site is unusually complex and located within a topologically closed hydrophilic beta-barrel (the triphosphate tunnel). Here we probe the active site of Plasmodium falciparum RNA Triphosphatase by targeted mutagenesis and thereby identify eight residues essential for catalysis. The functional data engender an improved structural alignment in which the Plasmodium counterparts of the Cet1 tunnel strands and active-site functional groups are located with confidence. We gain insight into the evolution of the Cet1-like Triphosphatase family by noting that the heretofore unique tertiary structure and active site of Cet1 are recapitulated in recently deposited structures of proteins from Pyrococcus (PBD 1YEM) and Vibrio (PDB 2ACA). The latter proteins exemplify a CYTH domain found in CyaB-like adenylate cyclases and mammalian thiamine Triphosphatase. We conclude that the tunnel fold first described for Cet1 is the prototype of a larger enzyme superfamily that includes the CYTH branch. This superfamily, which we name "triphosphate tunnel metalloenzyme," is distributed widely among bacterial, archaeal, and eukaryal taxa. It is now clear that Cet1-like RNA Triphosphatases did not arise de novo in unicellular eukarya in tandem with the emergence of caps as the defining feature of eukaryotic mRNA. They likely evolved by incremental changes in an ancestral tunnel enzyme that conferred specificity for RNA 5'-end processing.
-
Structure-Function Analysis of Trypanosoma brucei RNA Triphosphatase and Evidence for a Two-metal Mechanism
Journal of Biological Chemistry, 2003Co-Authors: Chunling Gong, Alexandra Martins, Stewart ShumanAbstract:Abstract Trypanosoma brucei RNA Triphosphatase TbCet1 is a 252-amino acid polypeptide that catalyzes the first step in mRNA cap formation. By performing an alanine scan of TbCet1, we identified six amino acids that are essential for Triphosphatase activity (Glu-52, Arg-127, Glu-168, Arg-186, Glu-216, and Glu-218). These results consolidate the proposal that protozoan, fungal, and Chlorella virus RNA Triphosphatases belong to a single family of metal-dependent NTP phosphohydrolases with a unique tunnel active site composed of eight β strands. Limited proteolysis of TbCet1 suggests that the hydrophilic N terminus is surface-exposed, whereas the catalytic core domain is tightly folded with the exception of a protease-sensitive loop (76WKGRRARKT84) between two of the putative tunnel strands. The catalytic domain of TbCet1 is extraordinarily thermostable. It remains active after heating for 2 h at 75 °C. Analysis by zonal velocity sedimentation indicates that TbCet1 is a monomeric enzyme, unlike fungal RNA Triphosphatases, which are homodimers. We show that tripolyphosphate is a potent competitive inhibitor of TbCet1 (Ki 1.4 μm) that binds more avidly to the active site than the ATP substrate (Km 25 μm). We present evidence of synergistic activation of the TbCet1 Triphosphatase by manganese and magnesium, consistent with a two-metal mechanism of catalysis. Our findings provide new insight to the similarities (in active site tertiary structure and catalytic mechanism) and differences (in quaternary structure and thermal stability) among the different branches of the tunnel enzyme family.
-
Homodimeric quaternary structure is required for the in vivo function and thermal stability of Saccharomyces cerevisiae and Schizosaccharomyces pombe RNA Triphosphatases.
Journal of Biological Chemistry, 2003Co-Authors: Stephane Hausmann, Stewart ShumanAbstract:Abstract Saccharomyces cerevisiae Cet1 and Schizosaccharomyces pombe Pct1 are the essential RNA Triphosphatase components of the mRNA capping apparatus of budding and fission yeast, respectively. Cet1 and Pct1 share a baroque active site architecture and a homodimeric quaternary structure. The active site is located within a topologically closed hydrophilic β-barrel (the triphosphate tunnel) that rests on a globular core domain (the pedestal) composed of elements from both protomers of the homodimer. Earlier studies of the effects of alanine cluster mutations at the crystallographic dimer interface of Cet1 suggested that homodimerization is important for Triphosphatase function in vivo, albeit not for catalysis. Here, we studied the effects of 14 single-alanine mutations on Cet1 activity and thereby pinpointed Asp280 as a critical side chain required for dimer formation. We find that disruption of the dimer interface is lethal in vivo and renders Cet1 activity thermolabile at physiological temperatures in vitro. In addition, we identify individual residues within the pedestal domain (Ile470, Leu519, Ile520, Phe523, Leu524, and Ile530) that stabilize Cet1 in vivo and in vitro. In the case of Pct1, we show that dimerization depends on the peptide segment 41VPKIEMNFLN50 located immediately prior to the start of the Pct1 catalytic domain. Deletion of this peptide converts Pct1 into a catalytically active monomer that is defective in vivo in S. pombe and hypersensitive to thermal inactivation in vitro. Our findings suggest an explanation for the conservation of quaternary structure in fungal RNA Triphosphatases, whereby the delicate tunnel architecture of the active site is stabilized by the homodimeric pedestal domain.
-
Mapping the active site of vaccinia virus RNA Triphosphatase.
Virology, 2003Co-Authors: Chunling Gong, Stewart ShumanAbstract:Abstract The RNA Triphosphatase component of vaccinia virus mRNA capping enzyme (the product of the viral D1 gene) belongs to a family of metal-dependent phosphohydrolases that includes the RNA Triphosphatases of fungi, protozoa, Chlorella virus, and baculoviruses. The family is defined by two glutamate-containing motifs (A and C) that form the metal-binding site. Most of the family members resemble the fungal and Chlorella virus enzymes, which have a complex active site located within the hydrophilic interior of a topologically closed eight-stranded β barrel (the so-called “triphosphate tunnel”). Here we queried whether vaccinia virus capping enzyme is a member of the tunnel subfamily, via mutational mapping of amino acids required for vaccinia Triphosphatase activity. We identified four new essential side chains in vaccinia D1 via alanine scanning and illuminated structure–activity relationships by conservative substitutions. Our results, together with previous mutational data, highlight a constellation of six acidic and three basic amino acids that likely compose the vaccinia Triphosphatase active site (Glu37, Glu39, Arg77, Lys107, Glu126, Asp159, Lys161, Glu192, and Glu194). These nine essential residues are conserved in all vertebrate and invertebrate poxvirus RNA capping enzymes. We discerned no pattern of clustering of the catalytic residues of the poxvirus Triphosphatase that would suggest structural similarity to the tunnel proteins (exclusive of motifs A and C). We infer that the poxvirus Triphosphatases are a distinct lineage within the metal-dependent RNA Triphosphatase family. Their unique active site, which is completely different from that of the host cell’s capping enzyme, recommends the poxvirus RNA Triphosphatase as a molecular target for antipoxviral drug discovery.
-
mapping the Triphosphatase active site of baculovirus mrna capping enzyme lef4 and evidence for a two metal mechanism
Nucleic Acids Research, 2003Co-Authors: Alexandra Martins, Stewart ShumanAbstract:The 464-amino acid baculovirus LEF4 protein is a bifunctional mRNA capping enzyme with Triphosphatase and guanylyltransferase activities. The N-terminal half of LEF4 constitutes an autonomous Triphosphatase catalytic domain. The LEF4 Triphosphatase belongs to a family of metal-dependent phosphohydrolases, which includes the RNA Triphosphatases of fungi, protozoa, Chlorella virus and poxviruses. The family is defined by two glutamate-containing motifs (A and C), which form a metal-binding site. Most of the family members resemble the fungal and Chlorella virus enzymes, which have a complex active site located within the hydrophilic interior of a topologically closed eight stranded beta barrel (the so-called 'triphosphate tunnel'). Here we probed whether baculovirus LEF4 is a member of the tunnel subfamily, via mutational mapping of amino acids required for Triphosphatase activity. We identified four new essential side chains in LEF4 via alanine scanning and illuminated structure-activity relationships by conservative substitutions. Our results, together with previous mutational data, highlight five acidic and four basic amino acids that are likely to comprise the LEF4 Triphosphatase active site (Glu9, Glu11, Arg51, Arg53, Glu97, Lys126, Arg179, Glu181 and Glu183). These nine essential residues are conserved in LEF4 orthologs from all strains of baculoviruses. We discerned no pattern of clustering of the catalytic residues of the baculovirus Triphosphatase that would suggest structural similarity to the tunnel proteins (exclusive of motifs A and C). However, there is similarity to the active site of vaccinia RNA Triphosphatase. We infer that the baculovirus and poxvirus Triphosphatases are a distinct lineage within the metal-dependent RNA Triphosphatase family. Synergistic activation of the LEF4 Triphosphatase by manganese and magnesium suggests a two-metal mechanism of gamma phosphate hydrolysis.
Radhakrishnan Padmanabhan - One of the best experts on this subject based on the ideXlab platform.
-
expression purification and characterization of the rna 5 Triphosphatase activity of dengue virus type 2 nonstructural protein 3
Virology, 2002Co-Authors: Greg Bartelma, Radhakrishnan PadmanabhanAbstract:Abstract Dengue virus type 2 (DEN2), a member of the Flaviviridae family of positive-strand RNA viruses, contains a single RNA genome having a type I cap structure at the 5′ end. The viral RNA is translated to produce a single polyprotein precursor that is processed to yield three virion proteins and at least seven nonstructural proteins (NS) in the infected host. NS3 is a multifunctional protein having a serine protease catalytic triad within the N-terminal 180 amino acid residues which requires NS2B as a cofactor for activation of protease activity. The C-terminal portion of this catalytic triad has conserved motifs present in several nucleoside Triphosphatases (NTPases)/RNA helicases. In addition, subtilisin-treated West Nile (WN) virus NS3 from infected cells was reported to have 5′-RNA Triphosphatase activity, suggesting its role in the synthesis of the 5′-cap structure. In this study, full-length DEN2 NS3 was expressed with an N-terminal histidine tag in Escherichia coli and purified in a soluble form. The purified protein has 5′-RNA Triphosphatase activity that cleaves the γ-phosphate moiety of the 5′-triphosphorylated RNA substrate. Biochemical and mutational analyses of the NS3 protein indicate that the nucleoside Triphosphatase and 5′-RNA Triphosphatase activities of NS3 share a common active site.
-
Expression, Purification, and Characterization of the RNA 5′-Triphosphatase Activity of Dengue Virus Type 2 Nonstructural Protein 3
Virology, 2002Co-Authors: Greg Bartelma, Radhakrishnan PadmanabhanAbstract:Abstract Dengue virus type 2 (DEN2), a member of the Flaviviridae family of positive-strand RNA viruses, contains a single RNA genome having a type I cap structure at the 5′ end. The viral RNA is translated to produce a single polyprotein precursor that is processed to yield three virion proteins and at least seven nonstructural proteins (NS) in the infected host. NS3 is a multifunctional protein having a serine protease catalytic triad within the N-terminal 180 amino acid residues which requires NS2B as a cofactor for activation of protease activity. The C-terminal portion of this catalytic triad has conserved motifs present in several nucleoside Triphosphatases (NTPases)/RNA helicases. In addition, subtilisin-treated West Nile (WN) virus NS3 from infected cells was reported to have 5′-RNA Triphosphatase activity, suggesting its role in the synthesis of the 5′-cap structure. In this study, full-length DEN2 NS3 was expressed with an N-terminal histidine tag in Escherichia coli and purified in a soluble form. The purified protein has 5′-RNA Triphosphatase activity that cleaves the γ-phosphate moiety of the 5′-triphosphorylated RNA substrate. Biochemical and mutational analyses of the NS3 protein indicate that the nucleoside Triphosphatase and 5′-RNA Triphosphatase activities of NS3 share a common active site.
Chunling Gong - One of the best experts on this subject based on the ideXlab platform.
-
Structure-function analysis of Plasmodium RNA Triphosphatase and description of a triphosphate tunnel metalloenzyme superfamily that includes Cet1-like RNA Triphosphatases and CYTH proteins.
RNA (New York N.Y.), 2006Co-Authors: Chunling Gong, Paul Smith, Stewart ShumanAbstract:RNA Triphosphatase catalyzes the first step in mRNA capping. The RNA Triphosphatases of fungi and protozoa are structurally and mechanistically unrelated to the analogous mammalian enzyme, a situation that recommends RNA Triphosphatase as an anti-infective target. Fungal and protozoan RNA Triphosphatases belong to a family of metal-dependent phosphohydrolases exemplified by yeast Cet1. The Cet1 active site is unusually complex and located within a topologically closed hydrophilic beta-barrel (the triphosphate tunnel). Here we probe the active site of Plasmodium falciparum RNA Triphosphatase by targeted mutagenesis and thereby identify eight residues essential for catalysis. The functional data engender an improved structural alignment in which the Plasmodium counterparts of the Cet1 tunnel strands and active-site functional groups are located with confidence. We gain insight into the evolution of the Cet1-like Triphosphatase family by noting that the heretofore unique tertiary structure and active site of Cet1 are recapitulated in recently deposited structures of proteins from Pyrococcus (PBD 1YEM) and Vibrio (PDB 2ACA). The latter proteins exemplify a CYTH domain found in CyaB-like adenylate cyclases and mammalian thiamine Triphosphatase. We conclude that the tunnel fold first described for Cet1 is the prototype of a larger enzyme superfamily that includes the CYTH branch. This superfamily, which we name "triphosphate tunnel metalloenzyme," is distributed widely among bacterial, archaeal, and eukaryal taxa. It is now clear that Cet1-like RNA Triphosphatases did not arise de novo in unicellular eukarya in tandem with the emergence of caps as the defining feature of eukaryotic mRNA. They likely evolved by incremental changes in an ancestral tunnel enzyme that conferred specificity for RNA 5'-end processing.
-
Structure-Function Analysis of Trypanosoma brucei RNA Triphosphatase and Evidence for a Two-metal Mechanism
Journal of Biological Chemistry, 2003Co-Authors: Chunling Gong, Alexandra Martins, Stewart ShumanAbstract:Abstract Trypanosoma brucei RNA Triphosphatase TbCet1 is a 252-amino acid polypeptide that catalyzes the first step in mRNA cap formation. By performing an alanine scan of TbCet1, we identified six amino acids that are essential for Triphosphatase activity (Glu-52, Arg-127, Glu-168, Arg-186, Glu-216, and Glu-218). These results consolidate the proposal that protozoan, fungal, and Chlorella virus RNA Triphosphatases belong to a single family of metal-dependent NTP phosphohydrolases with a unique tunnel active site composed of eight β strands. Limited proteolysis of TbCet1 suggests that the hydrophilic N terminus is surface-exposed, whereas the catalytic core domain is tightly folded with the exception of a protease-sensitive loop (76WKGRRARKT84) between two of the putative tunnel strands. The catalytic domain of TbCet1 is extraordinarily thermostable. It remains active after heating for 2 h at 75 °C. Analysis by zonal velocity sedimentation indicates that TbCet1 is a monomeric enzyme, unlike fungal RNA Triphosphatases, which are homodimers. We show that tripolyphosphate is a potent competitive inhibitor of TbCet1 (Ki 1.4 μm) that binds more avidly to the active site than the ATP substrate (Km 25 μm). We present evidence of synergistic activation of the TbCet1 Triphosphatase by manganese and magnesium, consistent with a two-metal mechanism of catalysis. Our findings provide new insight to the similarities (in active site tertiary structure and catalytic mechanism) and differences (in quaternary structure and thermal stability) among the different branches of the tunnel enzyme family.
-
Mapping the active site of vaccinia virus RNA Triphosphatase.
Virology, 2003Co-Authors: Chunling Gong, Stewart ShumanAbstract:Abstract The RNA Triphosphatase component of vaccinia virus mRNA capping enzyme (the product of the viral D1 gene) belongs to a family of metal-dependent phosphohydrolases that includes the RNA Triphosphatases of fungi, protozoa, Chlorella virus, and baculoviruses. The family is defined by two glutamate-containing motifs (A and C) that form the metal-binding site. Most of the family members resemble the fungal and Chlorella virus enzymes, which have a complex active site located within the hydrophilic interior of a topologically closed eight-stranded β barrel (the so-called “triphosphate tunnel”). Here we queried whether vaccinia virus capping enzyme is a member of the tunnel subfamily, via mutational mapping of amino acids required for vaccinia Triphosphatase activity. We identified four new essential side chains in vaccinia D1 via alanine scanning and illuminated structure–activity relationships by conservative substitutions. Our results, together with previous mutational data, highlight a constellation of six acidic and three basic amino acids that likely compose the vaccinia Triphosphatase active site (Glu37, Glu39, Arg77, Lys107, Glu126, Asp159, Lys161, Glu192, and Glu194). These nine essential residues are conserved in all vertebrate and invertebrate poxvirus RNA capping enzymes. We discerned no pattern of clustering of the catalytic residues of the poxvirus Triphosphatase that would suggest structural similarity to the tunnel proteins (exclusive of motifs A and C). We infer that the poxvirus Triphosphatases are a distinct lineage within the metal-dependent RNA Triphosphatase family. Their unique active site, which is completely different from that of the host cell’s capping enzyme, recommends the poxvirus RNA Triphosphatase as a molecular target for antipoxviral drug discovery.
Alexandra Martins - One of the best experts on this subject based on the ideXlab platform.
-
Structure-Function Analysis of Trypanosoma brucei RNA Triphosphatase and Evidence for a Two-metal Mechanism
Journal of Biological Chemistry, 2003Co-Authors: Chunling Gong, Alexandra Martins, Stewart ShumanAbstract:Abstract Trypanosoma brucei RNA Triphosphatase TbCet1 is a 252-amino acid polypeptide that catalyzes the first step in mRNA cap formation. By performing an alanine scan of TbCet1, we identified six amino acids that are essential for Triphosphatase activity (Glu-52, Arg-127, Glu-168, Arg-186, Glu-216, and Glu-218). These results consolidate the proposal that protozoan, fungal, and Chlorella virus RNA Triphosphatases belong to a single family of metal-dependent NTP phosphohydrolases with a unique tunnel active site composed of eight β strands. Limited proteolysis of TbCet1 suggests that the hydrophilic N terminus is surface-exposed, whereas the catalytic core domain is tightly folded with the exception of a protease-sensitive loop (76WKGRRARKT84) between two of the putative tunnel strands. The catalytic domain of TbCet1 is extraordinarily thermostable. It remains active after heating for 2 h at 75 °C. Analysis by zonal velocity sedimentation indicates that TbCet1 is a monomeric enzyme, unlike fungal RNA Triphosphatases, which are homodimers. We show that tripolyphosphate is a potent competitive inhibitor of TbCet1 (Ki 1.4 μm) that binds more avidly to the active site than the ATP substrate (Km 25 μm). We present evidence of synergistic activation of the TbCet1 Triphosphatase by manganese and magnesium, consistent with a two-metal mechanism of catalysis. Our findings provide new insight to the similarities (in active site tertiary structure and catalytic mechanism) and differences (in quaternary structure and thermal stability) among the different branches of the tunnel enzyme family.
-
mapping the Triphosphatase active site of baculovirus mrna capping enzyme lef4 and evidence for a two metal mechanism
Nucleic Acids Research, 2003Co-Authors: Alexandra Martins, Stewart ShumanAbstract:The 464-amino acid baculovirus LEF4 protein is a bifunctional mRNA capping enzyme with Triphosphatase and guanylyltransferase activities. The N-terminal half of LEF4 constitutes an autonomous Triphosphatase catalytic domain. The LEF4 Triphosphatase belongs to a family of metal-dependent phosphohydrolases, which includes the RNA Triphosphatases of fungi, protozoa, Chlorella virus and poxviruses. The family is defined by two glutamate-containing motifs (A and C), which form a metal-binding site. Most of the family members resemble the fungal and Chlorella virus enzymes, which have a complex active site located within the hydrophilic interior of a topologically closed eight stranded beta barrel (the so-called 'triphosphate tunnel'). Here we probed whether baculovirus LEF4 is a member of the tunnel subfamily, via mutational mapping of amino acids required for Triphosphatase activity. We identified four new essential side chains in LEF4 via alanine scanning and illuminated structure-activity relationships by conservative substitutions. Our results, together with previous mutational data, highlight five acidic and four basic amino acids that are likely to comprise the LEF4 Triphosphatase active site (Glu9, Glu11, Arg51, Arg53, Glu97, Lys126, Arg179, Glu181 and Glu183). These nine essential residues are conserved in LEF4 orthologs from all strains of baculoviruses. We discerned no pattern of clustering of the catalytic residues of the baculovirus Triphosphatase that would suggest structural similarity to the tunnel proteins (exclusive of motifs A and C). However, there is similarity to the active site of vaccinia RNA Triphosphatase. We infer that the baculovirus and poxvirus Triphosphatases are a distinct lineage within the metal-dependent RNA Triphosphatase family. Synergistic activation of the LEF4 Triphosphatase by manganese and magnesium suggests a two-metal mechanism of gamma phosphate hydrolysis.
-
Mapping the Triphosphatase active site of baculovirus mRNA capping enzyme LEF4 and evidence for a two‐metal mechanism
Nucleic acids research, 2003Co-Authors: Alexandra Martins, Stewart ShumanAbstract:The 464-amino acid baculovirus LEF4 protein is a bifunctional mRNA capping enzyme with Triphosphatase and guanylyltransferase activities. The N-terminal half of LEF4 constitutes an autonomous Triphosphatase catalytic domain. The LEF4 Triphosphatase belongs to a family of metal-dependent phosphohydrolases, which includes the RNA Triphosphatases of fungi, protozoa, Chlorella virus and poxviruses. The family is defined by two glutamate-containing motifs (A and C), which form a metal-binding site. Most of the family members resemble the fungal and Chlorella virus enzymes, which have a complex active site located within the hydrophilic interior of a topologically closed eight stranded beta barrel (the so-called 'triphosphate tunnel'). Here we probed whether baculovirus LEF4 is a member of the tunnel subfamily, via mutational mapping of amino acids required for Triphosphatase activity. We identified four new essential side chains in LEF4 via alanine scanning and illuminated structure-activity relationships by conservative substitutions. Our results, together with previous mutational data, highlight five acidic and four basic amino acids that are likely to comprise the LEF4 Triphosphatase active site (Glu9, Glu11, Arg51, Arg53, Glu97, Lys126, Arg179, Glu181 and Glu183). These nine essential residues are conserved in LEF4 orthologs from all strains of baculoviruses. We discerned no pattern of clustering of the catalytic residues of the baculovirus Triphosphatase that would suggest structural similarity to the tunnel proteins (exclusive of motifs A and C). However, there is similarity to the active site of vaccinia RNA Triphosphatase. We infer that the baculovirus and poxvirus Triphosphatases are a distinct lineage within the metal-dependent RNA Triphosphatase family. Synergistic activation of the LEF4 Triphosphatase by manganese and magnesium suggests a two-metal mechanism of gamma phosphate hydrolysis.
-
mutational analysis of baculovirus capping enzyme lef4 delineates an autonomous Triphosphatase domain and structural determinants of divalent cation specificity
Journal of Biological Chemistry, 2001Co-Authors: Alexandra Martins, Stewart ShumanAbstract:Abstract The 464-amino acid baculovirus Lef4 protein is a bifunctional mRNA capping enzyme with Triphosphatase and guanylyltransferase activities. The hydrolysis of 5′-triphosphate RNA and free NTPs by Lef4 is dependent on a divalent cation cofactor. RNA Triphosphatase activity is optimal at pH 7.5 with either magnesium or manganese, yet NTP hydrolysis at neutral pH is activated only by manganese or cobalt. Here we show that Lef4 possesses an intrinsic magnesium-dependent ATPase with a distinctive alkaline pH optimum and a high K m for ATP (4 mm). Lef4 contains two conserved sequences, motif A (8IEKEISY14) and motif C (180LEYEF184), which define the fungal/viral/protozoal family of metal-dependent RNA Triphosphatases. We find by mutational analysis that Glu9, Glu11, Glu181, and Glu183 are essential for phosphohydrolase chemistry and likely comprise the metal-binding site of Lef4. Conservative mutations E9D and E183D abrogate the magnesium-dependent Triphosphatase activities of Lef4 and transform it into a strictly manganese-dependent RNA Triphosphatase. Limited proteolysis of Lef4 and ensuing COOH-terminal deletion analysis revealed that the NH2-terminal 236-amino acid segment of Lef4 constitutes an autonomous Triphosphatase catalytic domain.
Greg Bartelma - One of the best experts on this subject based on the ideXlab platform.
-
expression purification and characterization of the rna 5 Triphosphatase activity of dengue virus type 2 nonstructural protein 3
Virology, 2002Co-Authors: Greg Bartelma, Radhakrishnan PadmanabhanAbstract:Abstract Dengue virus type 2 (DEN2), a member of the Flaviviridae family of positive-strand RNA viruses, contains a single RNA genome having a type I cap structure at the 5′ end. The viral RNA is translated to produce a single polyprotein precursor that is processed to yield three virion proteins and at least seven nonstructural proteins (NS) in the infected host. NS3 is a multifunctional protein having a serine protease catalytic triad within the N-terminal 180 amino acid residues which requires NS2B as a cofactor for activation of protease activity. The C-terminal portion of this catalytic triad has conserved motifs present in several nucleoside Triphosphatases (NTPases)/RNA helicases. In addition, subtilisin-treated West Nile (WN) virus NS3 from infected cells was reported to have 5′-RNA Triphosphatase activity, suggesting its role in the synthesis of the 5′-cap structure. In this study, full-length DEN2 NS3 was expressed with an N-terminal histidine tag in Escherichia coli and purified in a soluble form. The purified protein has 5′-RNA Triphosphatase activity that cleaves the γ-phosphate moiety of the 5′-triphosphorylated RNA substrate. Biochemical and mutational analyses of the NS3 protein indicate that the nucleoside Triphosphatase and 5′-RNA Triphosphatase activities of NS3 share a common active site.
-
Expression, Purification, and Characterization of the RNA 5′-Triphosphatase Activity of Dengue Virus Type 2 Nonstructural Protein 3
Virology, 2002Co-Authors: Greg Bartelma, Radhakrishnan PadmanabhanAbstract:Abstract Dengue virus type 2 (DEN2), a member of the Flaviviridae family of positive-strand RNA viruses, contains a single RNA genome having a type I cap structure at the 5′ end. The viral RNA is translated to produce a single polyprotein precursor that is processed to yield three virion proteins and at least seven nonstructural proteins (NS) in the infected host. NS3 is a multifunctional protein having a serine protease catalytic triad within the N-terminal 180 amino acid residues which requires NS2B as a cofactor for activation of protease activity. The C-terminal portion of this catalytic triad has conserved motifs present in several nucleoside Triphosphatases (NTPases)/RNA helicases. In addition, subtilisin-treated West Nile (WN) virus NS3 from infected cells was reported to have 5′-RNA Triphosphatase activity, suggesting its role in the synthesis of the 5′-cap structure. In this study, full-length DEN2 NS3 was expressed with an N-terminal histidine tag in Escherichia coli and purified in a soluble form. The purified protein has 5′-RNA Triphosphatase activity that cleaves the γ-phosphate moiety of the 5′-triphosphorylated RNA substrate. Biochemical and mutational analyses of the NS3 protein indicate that the nucleoside Triphosphatase and 5′-RNA Triphosphatase activities of NS3 share a common active site.
