The Experts below are selected from a list of 258 Experts worldwide ranked by ideXlab platform
Kiyotaka Hitomi - One of the best experts on this subject based on the ideXlab platform.
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Identification of preferred substrate sequences of microbial transglutaminase from Streptomyces mobaraensis using a phage-displayed peptide library
Archives of Biochemistry and Biophysics, 2008Co-Authors: Yoshiaki Sugimura, Masatoshi Maki, Keiichi Yokoyama, Kiyotaka HitomiAbstract:Microbial transglutaminase (TGase) from Streptomyces mobaraensis (MTG) has been used in many industrial applications because it effectively catalyzes the formation of covalent cross-linking between Glutamine Residues in various substrate proteins and lysine Residues or primary amines. To better understand the sequence preference around the reactive Glutamine Residue by this enzymatic reaction, we screened preferred peptide sequences using a phage-displayed random peptide library. Most of the peptides identified contained a consensus sequence, which was different from those previously found for mammalian TGases. Of these, most sequences had a specific reactivity toward MTG when produced as a fusion protein with glutathione-S-transferase. Furthermore, the representative sequence was found to be reactive even in the peptide form. The amino acid Residues in the sequence critical for the reactivity were further analyzed, and the possible interaction with the enzyme has been discussed in this paper.
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Screening for the preferred substrate sequence of transglutaminase using a phage-displayed peptide library: Identification of peptide substrates for TGase 2 and factor XIIIa
Journal of Biological Chemistry, 2006Co-Authors: Yoshiaki Sugimura, Masayo Hosono, Fumitaka Wada, Masatoshi Maki, Tohru Yoshimura, Kiyotaka HitomiAbstract:Mammalian transglutaminase (TGase) catalyzes covalent cross-linking of peptide-bound lysine Residues or incorporation of primary amines to limited Glutamine Residues in substrate proteins. Using an unbiased M13 phage display random peptide library, we developed a screening system to elucidate primary structures surrounding reactive Glutamine Residue(s) that are preferred by TGase. Screening was performed by selecting phage clones expressing peptides that incorporated biotin-labeled primary amine by the catalytic reactions of TGase 2 and activated Factor XIII ( Factor XIIIa). We identified several amino acid sequences that were preferred as Glutamine donor substrates, most of which have a marked tendency for individual TGases: TGase 2, QxP phi D( P), QxP phi, and Qxx phi DP; Factor XIIIa, Qxx phi xWP (where x and phi represent a non-conserved and a hydrophobic amino acid, respectively). We further confirmed that the sequences were favored for transamidation using modified glutathione S-transferase (GST) for recombinant peptide-GST fusion proteins. Most of the fusion proteins exhibited a considerable increase in incorporation of primary amines over that of modified GST alone. Furthermore, we identified the amino acid sequences that demonstrated higher specificity and inhibitory activity in the cross-linking reactions by TGase 2 and Factor XIIIa.
Yoshiaki Sugimura - One of the best experts on this subject based on the ideXlab platform.
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Identification of preferred substrate sequences of microbial transglutaminase from Streptomyces mobaraensis using a phage-displayed peptide library
Archives of Biochemistry and Biophysics, 2008Co-Authors: Yoshiaki Sugimura, Masatoshi Maki, Keiichi Yokoyama, Kiyotaka HitomiAbstract:Microbial transglutaminase (TGase) from Streptomyces mobaraensis (MTG) has been used in many industrial applications because it effectively catalyzes the formation of covalent cross-linking between Glutamine Residues in various substrate proteins and lysine Residues or primary amines. To better understand the sequence preference around the reactive Glutamine Residue by this enzymatic reaction, we screened preferred peptide sequences using a phage-displayed random peptide library. Most of the peptides identified contained a consensus sequence, which was different from those previously found for mammalian TGases. Of these, most sequences had a specific reactivity toward MTG when produced as a fusion protein with glutathione-S-transferase. Furthermore, the representative sequence was found to be reactive even in the peptide form. The amino acid Residues in the sequence critical for the reactivity were further analyzed, and the possible interaction with the enzyme has been discussed in this paper.
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Screening for the preferred substrate sequence of transglutaminase using a phage-displayed peptide library: Identification of peptide substrates for TGase 2 and factor XIIIa
Journal of Biological Chemistry, 2006Co-Authors: Yoshiaki Sugimura, Masayo Hosono, Fumitaka Wada, Masatoshi Maki, Tohru Yoshimura, Kiyotaka HitomiAbstract:Mammalian transglutaminase (TGase) catalyzes covalent cross-linking of peptide-bound lysine Residues or incorporation of primary amines to limited Glutamine Residues in substrate proteins. Using an unbiased M13 phage display random peptide library, we developed a screening system to elucidate primary structures surrounding reactive Glutamine Residue(s) that are preferred by TGase. Screening was performed by selecting phage clones expressing peptides that incorporated biotin-labeled primary amine by the catalytic reactions of TGase 2 and activated Factor XIII ( Factor XIIIa). We identified several amino acid sequences that were preferred as Glutamine donor substrates, most of which have a marked tendency for individual TGases: TGase 2, QxP phi D( P), QxP phi, and Qxx phi DP; Factor XIIIa, Qxx phi xWP (where x and phi represent a non-conserved and a hydrophobic amino acid, respectively). We further confirmed that the sequences were favored for transamidation using modified glutathione S-transferase (GST) for recombinant peptide-GST fusion proteins. Most of the fusion proteins exhibited a considerable increase in incorporation of primary amines over that of modified GST alone. Furthermore, we identified the amino acid sequences that demonstrated higher specificity and inhibitory activity in the cross-linking reactions by TGase 2 and Factor XIIIa.
Koichiro Ishimori - One of the best experts on this subject based on the ideXlab platform.
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unique peroxidase reaction mechanism in prostaglandin endoperoxide h synthase 2 compound i in prostaglandin endoperoxide h synthase 2 can be formed without assistance by distal Glutamine Residue
Journal of Biological Chemistry, 2007Co-Authors: Shizuo Ichimura, Takeshi Uchida, Shuhei Taniguchi, Shusuke Hira, Takehiko Tosha, Isao Morishima, Teizo Kitagawa, Koichiro IshimoriAbstract:Abstract Prostaglandin-endoperoxide H synthase-2 (PGHS-2) shows peroxidase activity to promote the cyclooxygenase reaction for prostaglandin H2, but one of the highly conserved amino acid Residues in peroxidases, distal Arg, stabilizing the developing negative charge on the peroxide through a hydrogen-bonding interaction, is replaced with a neutral amino acid Residue, Gln. To characterize the peroxidase reaction in PGHS-2, we prepared three distal Glutamine (Gln-189) mutants, Arg (Gln→Arg), Asn (Gln→ Asn), and Val (Gln→ Val) mutants, and examined their peroxidase activity together with their structural characterization by absorption and resonance Raman spectra. Although a previous study (Landino, L. M., Crews, B. C., Gierse, J. K., Hauser, S. D., and Marnett, L. (1997) J. Biol. Chem. 272, 21565-21574) suggested that the Gln Residue might serve as a functionally equivalent Residue to Arg, our current results clearly showed that the peroxidase activity of the Val and Asn mutants was comparable with that of the wild-type enzyme. In addition, the Fe-C and C-O stretching modes in the CO adduct were almost unperturbed by the mutation, implying that Gln-189 might not directly interact with the heme-ligated peroxide. Rather, the peroxidase activity of the Arg mutant was depressed, concomitant with the heme environmental change from a six-coordinate to a five-coordinate structure. Introduction of the bulky amino acid Residue, Arg, would interfere with the ligation of a water molecule to the heme iron, suggesting that the side chain volume, and not the amide group, at position 189 is essential for the peroxidase activity of PGHS-2. Thus, we can conclude that the O-O bond cleavage in PGHS-2 is promoted without interactions with charged side chains at the peroxide binding site, which is significantly different from that in typical plant peroxidases.
Richard H Buckingham - One of the best experts on this subject based on the ideXlab platform.
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the Glutamine Residue of the conserved ggq motif in saccharomyces cerevisiae release factor erf1 is methylated by the product of the ydr140w gene
Journal of Biological Chemistry, 2005Co-Authors: Valerie Heurguehamard, L L Kisselev, Stephanie Champ, Liliana Mora, Tatiana Merkoulovarainon, Richard H BuckinghamAbstract:Abstract Polypeptide release factors from eubacteria and eukaryotes, although similar in function, belong to different protein families. They share one sequence motif, a GGQ tripeptide that is vital to release factor (RF) activity in both kingdoms. In bacteria, the Gln Residue of the motif in RF1 and RF2 is modified to N5-methyl-Gln by the S-adenosyl l-methionine-dependent methyltransferase PrmC and the absence of Gln methylation decreases the release activity of Escherichia coli RF2 in vitro severalfold. We show here that the same modification is made to the GGQ motif of Saccharomyces cerevisiae release factor eRF1, the first time that N5-methyl-Gln has been found outside the bacterial kingdom. The product of the YDR140w gene is required for the methylation of eRF1 in vivo and for optimal yeast cell growth. YDR140w protein has significant homology to PrmC but lacks the N-terminal domain thought to be involved in the recognition of the bacterial release factors. Overproduced in S. cerevisiae, YDR140w can methylate eRF1 from yeast or man in vitro using S-adenosyl l-methionine as methyl donor provided that eRF3 and GTP are also present, suggesting that the natural substrate of the methyltransferase YDR140w is the ternary complex eRF1·eRF3·GTP.
Gary J Hunter - One of the best experts on this subject based on the ideXlab platform.
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thermostability of manganese and iron superoxide dismutases from escherichia coli is determined by the characteristic position of a Glutamine Residue
FEBS Journal, 2002Co-Authors: Tha Ra Se Hunter, Joe V Bannister, Gary J HunterAbstract:The structurally homologous mononuclear iron and manganese superoxide dismutases (FeSOD and MnSOD, respectively) contain a highly conserved Glutamine Residue in the active site which projects toward the active-site metal centre and participates in an extensive hydrogen bonding network. The position of this Residue is different for each SOD isoenzyme (Q69 in FeSOD and Q146 in MnSOD of Escherichia coli). Although site-directed mutant enzymes lacking this Glutamine Residue (FeSOD[Q69G] and MnSOD[Q146A]) demonstrated a higher degree of selectivity for their respective metal, they showed little or no activity compared with wild types. FeSOD double mutants (FeSOD[Q69G/A141Q]), which mimic the Glutamine position in MnSOD, elicited 25% the activity of wild-type FeSOD while the activity of the corresponding MnSOD double mutant (MnSOD[G77Q/Q146A]) increased to 150% (relative to wild-type MnSOD). Both double mutants showed reduced selectivity toward their metal. Differences exhibited in the thermostability of SOD activity was most obvious in the mutants that contained two Glutamine Residues (FeSOD[A141Q] and MnSOD[G77Q]), where the MnSOD mutant was thermostable and the FeSOD mutant was thermolabile. Significantly, the MnSOD double mutant exhibited a thermal-inactivation profile similar to that of wild-type FeSOD while that of the FeSOD double mutant was similar to wild-type MnSOD. We conclude therefore that the position of this Glutamine Residue contributes to metal selectivity and is responsible for some of the different physicochemical properties of these SODs, and in particular their characteristic thermostability.
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Thermostability of manganese‐ and iron‐superoxide dismutases from Escherichia coli is determined by the characteristic position of a Glutamine Residue
FEBS Journal, 2002Co-Authors: Thérèse Hunter, Joe V Bannister, Gary J HunterAbstract:The structurally homologous mononuclear iron and manganese superoxide dismutases (FeSOD and MnSOD, respectively) contain a highly conserved Glutamine Residue in the active site which projects toward the active-site metal centre and participates in an extensive hydrogen bonding network. The position of this Residue is different for each SOD isoenzyme (Q69 in FeSOD and Q146 in MnSOD of Escherichia coli). Although site-directed mutant enzymes lacking this Glutamine Residue (FeSOD[Q69G] and MnSOD[Q146A]) demonstrated a higher degree of selectivity for their respective metal, they showed little or no activity compared with wild types. FeSOD double mutants (FeSOD[Q69G/A141Q]), which mimic the Glutamine position in MnSOD, elicited 25% the activity of wild-type FeSOD while the activity of the corresponding MnSOD double mutant (MnSOD[G77Q/Q146A]) increased to 150% (relative to wild-type MnSOD). Both double mutants showed reduced selectivity toward their metal. Differences exhibited in the thermostability of SOD activity was most obvious in the mutants that contained two Glutamine Residues (FeSOD[A141Q] and MnSOD[G77Q]), where the MnSOD mutant was thermostable and the FeSOD mutant was thermolabile. Significantly, the MnSOD double mutant exhibited a thermal-inactivation profile similar to that of wild-type FeSOD while that of the FeSOD double mutant was similar to wild-type MnSOD. We conclude therefore that the position of this Glutamine Residue contributes to metal selectivity and is responsible for some of the different physicochemical properties of these SODs, and in particular their characteristic thermostability.