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Makoto Ito - One of the best experts on this subject based on the ideXlab platform.
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Molecular Cloning and catalytic mechanism of a novel glycosphingolipid degrading β n acetylgalactosaminidase from paenibacillus sp ts12
Journal of Biological Chemistry, 2011Co-Authors: Tomomi Sumida, Ken Fujimoto, Makoto ItoAbstract:Abstract We report here the Molecular Cloning, characterization, and catalytic mechanism of a novel glycosphingolipid-degrading β-N-acetylgalactosaminidase (β-NGA) from Paenibacillus sp. TS12 (NgaP). Consisting of 1034 putative amino acid residues, NgaP shares no sequence similarity with known proteins. Recombinant NgaP, expressed in Escherichia coli, cleaved the nonreducing terminal β-GalNAc residues of gangliotriaosylceramide and globotetraosylceramide. The enzyme hydrolyzed para-nitrophenyl-β-N-acetylgalactosaminide ∼100 times faster than para-nitrophenyl-β-N-acetylglucosaminide. GalNAc thiazoline, an analog of the oxazolinium intermediate and potent inhibitor for enzymes adopting substrate-assisted catalysis, competitively inhibited the enzyme. The Ki of the enzyme for GalNAc thiazoline was 1.3 nm, whereas that for GlcNAc thiazoline was 46.8 μm. Comparison of the secondary structure with those of known enzymes exhibiting substrate-assisted catalysis and point mutation analysis indicated that NgaP adopts substrate-assisted catalysis in which Glu-608 and Asp-607 could function as a proton donor and a stabilizer of the 2-acetamide group of the β-GalNAc at the active site, respectively. These results clearly indicate that NgaP is a β-NGA showing substrate-assisted catalysis. This is the first report describing the Molecular Cloning of a β-NGA adopting substrate-assisted catalysis.
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Molecular Cloning and crystal structural analysis of a novel beta-N-acetylhexosaminidase from Paenibacillus sp. TS12 capable of degrading glycosphingolipids.
Journal of Molecular Biology, 2009Co-Authors: Tomomi Sumida, Ryohei Ishii, Tatsuo Yanagisawa, Shigeyuki Yokoyama, Makoto ItoAbstract:We report the Molecular Cloning and characterization of two novel β-N-acetylhexosaminidases (β-HEX, EC 3.2.1.52) from Paenibacillus sp. strain TS12. The two β-HEXs (Hex1 and Hex2) were 70% identical in primary structure, and the N-terminal region of both enzymes showed significant similarity with β-HEXs belonging to glycoside hydrolase family 20 (GH20). Interestingly, however, the C-terminal region of Hex1 and Hex2 shared no sequence similarity with the GH20 β-HEXs or other known proteins. Both recombinant enzymes, expressed in Escherichia coli BL21(DE3), hydrolyzed the β-N-acetylhexosamine linkage of chitooligosaccharides and glycosphingolipids such as asialo GM2 and Gb4Cer in the absence of detergent. However, the enzyme was not able to hydrolyze GM2 ganglioside in the presence or in the absence of detergent. We determined three crystal structures of Hex1; the Hex1 deletion mutant Hex1-ΔC at a resolution of 1.8 A; Hex1-ΔC in complex with β-N-acetylglucosamine at 1.6 A; and Hex1-ΔC in complex with β-N-acetylgalactosamine at 1.9 A. We made a docking model of Hex1-ΔC with GM2 oligosaccharide, revealing that the sialic acid residue of GM2 could hinder access of the substrate to the active site cavity. This is the first report describing the Molecular Cloning, characterization and X-ray structure of a procaryotic β-HEX capable of hydrolyzing glycosphingolipids.
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Molecular Cloning and characterization of a novel glucocerebrosidase of Paenibacillus sp. TS12.
Journal of biochemistry, 2002Co-Authors: Tomomi Sumida, Noriyuki Sueyoshi, Makoto ItoAbstract:We report here the Molecular Cloning and characterization of a glucocerebrosidase [EC 3.2.1.45] from Paenibacillus sp. TS12. The open reading frame of the glucocerebrosidase gene consisted of 2,493 bp nucleotides and encoded 831 amino acid residues. The enzyme exhibited no sequence similarity with a classical glucocerebrosidase belonging to glycoside hydrolase (GH) family 30, but rather showed significant similarity with GH family 3 beta-glucosidases from Clostridium thermocellum, Ruminococcus albus, and Aspergillus aculeateus. The recombinant enzyme, expressed in Escherichia coli BL21(DE3)pLysS, had a Molecular weight of 90.7 kDa and hydrolyzed NBD-labeled glucosylceramide, but not galactosylceramide, GM1a or sphingomyelin. The enzyme was most active at pH 6.5, and its apparent Km and Vmax values for NBD-labeled glucosylceramide and p-nitrophenyl-beta-glucopyranoside were 223 microM and 1.60 micromol/min/mg of protein, and 593 microM and 112 micromol/min/mg of protein, respectively. Site-directed mutagenesis indicated that Asp-223 is an essential amino acid for the catalytic reaction and possibly functions a catalytic nucleophile, as in GH family 3 beta-glucosidases. This is the first report of the Molecular Cloning and characterization of a glucocerebrosidase from a procaryote.
Tomomi Sumida - One of the best experts on this subject based on the ideXlab platform.
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Molecular Cloning and catalytic mechanism of a novel glycosphingolipid degrading β n acetylgalactosaminidase from paenibacillus sp ts12
Journal of Biological Chemistry, 2011Co-Authors: Tomomi Sumida, Ken Fujimoto, Makoto ItoAbstract:Abstract We report here the Molecular Cloning, characterization, and catalytic mechanism of a novel glycosphingolipid-degrading β-N-acetylgalactosaminidase (β-NGA) from Paenibacillus sp. TS12 (NgaP). Consisting of 1034 putative amino acid residues, NgaP shares no sequence similarity with known proteins. Recombinant NgaP, expressed in Escherichia coli, cleaved the nonreducing terminal β-GalNAc residues of gangliotriaosylceramide and globotetraosylceramide. The enzyme hydrolyzed para-nitrophenyl-β-N-acetylgalactosaminide ∼100 times faster than para-nitrophenyl-β-N-acetylglucosaminide. GalNAc thiazoline, an analog of the oxazolinium intermediate and potent inhibitor for enzymes adopting substrate-assisted catalysis, competitively inhibited the enzyme. The Ki of the enzyme for GalNAc thiazoline was 1.3 nm, whereas that for GlcNAc thiazoline was 46.8 μm. Comparison of the secondary structure with those of known enzymes exhibiting substrate-assisted catalysis and point mutation analysis indicated that NgaP adopts substrate-assisted catalysis in which Glu-608 and Asp-607 could function as a proton donor and a stabilizer of the 2-acetamide group of the β-GalNAc at the active site, respectively. These results clearly indicate that NgaP is a β-NGA showing substrate-assisted catalysis. This is the first report describing the Molecular Cloning of a β-NGA adopting substrate-assisted catalysis.
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Molecular Cloning and crystal structural analysis of a novel beta-N-acetylhexosaminidase from Paenibacillus sp. TS12 capable of degrading glycosphingolipids.
Journal of Molecular Biology, 2009Co-Authors: Tomomi Sumida, Ryohei Ishii, Tatsuo Yanagisawa, Shigeyuki Yokoyama, Makoto ItoAbstract:We report the Molecular Cloning and characterization of two novel β-N-acetylhexosaminidases (β-HEX, EC 3.2.1.52) from Paenibacillus sp. strain TS12. The two β-HEXs (Hex1 and Hex2) were 70% identical in primary structure, and the N-terminal region of both enzymes showed significant similarity with β-HEXs belonging to glycoside hydrolase family 20 (GH20). Interestingly, however, the C-terminal region of Hex1 and Hex2 shared no sequence similarity with the GH20 β-HEXs or other known proteins. Both recombinant enzymes, expressed in Escherichia coli BL21(DE3), hydrolyzed the β-N-acetylhexosamine linkage of chitooligosaccharides and glycosphingolipids such as asialo GM2 and Gb4Cer in the absence of detergent. However, the enzyme was not able to hydrolyze GM2 ganglioside in the presence or in the absence of detergent. We determined three crystal structures of Hex1; the Hex1 deletion mutant Hex1-ΔC at a resolution of 1.8 A; Hex1-ΔC in complex with β-N-acetylglucosamine at 1.6 A; and Hex1-ΔC in complex with β-N-acetylgalactosamine at 1.9 A. We made a docking model of Hex1-ΔC with GM2 oligosaccharide, revealing that the sialic acid residue of GM2 could hinder access of the substrate to the active site cavity. This is the first report describing the Molecular Cloning, characterization and X-ray structure of a procaryotic β-HEX capable of hydrolyzing glycosphingolipids.
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Molecular Cloning and characterization of a novel glucocerebrosidase of Paenibacillus sp. TS12.
Journal of biochemistry, 2002Co-Authors: Tomomi Sumida, Noriyuki Sueyoshi, Makoto ItoAbstract:We report here the Molecular Cloning and characterization of a glucocerebrosidase [EC 3.2.1.45] from Paenibacillus sp. TS12. The open reading frame of the glucocerebrosidase gene consisted of 2,493 bp nucleotides and encoded 831 amino acid residues. The enzyme exhibited no sequence similarity with a classical glucocerebrosidase belonging to glycoside hydrolase (GH) family 30, but rather showed significant similarity with GH family 3 beta-glucosidases from Clostridium thermocellum, Ruminococcus albus, and Aspergillus aculeateus. The recombinant enzyme, expressed in Escherichia coli BL21(DE3)pLysS, had a Molecular weight of 90.7 kDa and hydrolyzed NBD-labeled glucosylceramide, but not galactosylceramide, GM1a or sphingomyelin. The enzyme was most active at pH 6.5, and its apparent Km and Vmax values for NBD-labeled glucosylceramide and p-nitrophenyl-beta-glucopyranoside were 223 microM and 1.60 micromol/min/mg of protein, and 593 microM and 112 micromol/min/mg of protein, respectively. Site-directed mutagenesis indicated that Asp-223 is an essential amino acid for the catalytic reaction and possibly functions a catalytic nucleophile, as in GH family 3 beta-glucosidases. This is the first report of the Molecular Cloning and characterization of a glucocerebrosidase from a procaryote.
S. Stones-havas - One of the best experts on this subject based on the ideXlab platform.
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A Practical Evaluation of Next Generation Sequencing & Molecular Cloning Software.
Journal of biomolecular techniques, 2013Co-Authors: K. Qaadri, R. Moir, M. Kearse, S. Buxton, A. Wilson, M. Cheung, B. Milicevic, W. Hengjie, J. Kuhn, S. Stones-havasAbstract:Laboratories using Next Generation Sequencing (NGS) technologies and/ or high-throughput Molecular Cloning experiments can spend a significant amount of their research budget on data analysis and data management. The decision to develop in-house software, to rely on combinations of free software packages, or to purchase commercial software can significantly affect productivity and ROI. In this talk, we will describe a practical software evaluation process that was developed to assist core facility managers and principal investigators in determining the best tools for DNA/RNA/protein sequence analysis and Molecular Cloning. Eleven software packages were evaluated using six criteria: interface design, data management, data analysis, feature availability, extensibility, and support. This evaluation recommends software packages that excel within each of the individual criteria and the overall best software package for sequence analysis & Molecular Cloning.
Javier García-nafría - One of the best experts on this subject based on the ideXlab platform.
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In vivo DNA assembly using common laboratory bacteria: A re-emerging tool to simplify Molecular Cloning
Journal of Biological Chemistry, 2019Co-Authors: Jake F. Watson, Javier García-nafríaAbstract:: Molecular Cloning is a cornerstone of biomedical, biotechnological, and synthetic biology research. As such, improved Cloning methodologies can significantly advance the speed and cost of research projects. Whereas current popular Cloning approaches use in vitro assembly of DNA fragments, in vivo Cloning offers potential for greater simplification. It is generally assumed that bacterial in vivo Cloning requires Escherichia coli strains with enhanced recombination ability; however, this is incorrect. A widely present, bacterial RecA-independent recombination pathway is re-emerging as a powerful tool for Molecular Cloning and DNA assembly. This poorly understood pathway offers optimal Cloning properties (i.e. seamless, directional, and sequence-independent) without requiring in vitro DNA assembly or specialized bacteria, therefore vastly simplifying Cloning procedures. Although the use of this pathway to perform DNA assembly was first reported over 25 years ago, it failed to gain popularity, possibly due to both technical and circumstantial reasons. Technical limitations have now been overcome, and recent reports have demonstrated its versatility for DNA manipulation. Here, we summarize the historical trajectory of this approach and collate recent reports to provide a roadmap for its optimal use. Given the simplified protocols and minimal requirements, Cloning using in vivo DNA assembly in E. coli has the potential to become widely employed across the Molecular biology community.
Alain E. Lagarde - One of the best experts on this subject based on the ideXlab platform.
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DNA Microarrays: A Molecular Cloning Manual.
The American Journal of Human Genetics, 2003Co-Authors: Alain E. LagardeAbstract:This first edition of the book on DNA microarrays is a welcome addition to a series of laboratory manuals published by the Cold Spring Harbor Laboratory Press over the past two decades. It perpetuates a tradition of practical laboratory guides, which started with the famous guide by Maniatis on Molecular Cloning in the mid-80s, followed by volumes dedicated to other subjects, such as Antibodies (1988), Genome Analysis (1997), and several model organisms.
