Crystallogr

Crystallogr. specific to -primeverosides, highly glycone-specific for 6-= 140 and 26 m, respectively (20). The monosaccharide glycoside analogue, -glucosylamidine, has no inhibitory activity at 500 m, consistent with the fact that PD barely hydrolyzes -d-glucopyranosides. Therefore, x-ray crystal analysis of PD in complex with (?)59.6, 88.8, 195.260.0, 88.2, 195.659.1, 89.9, 195.2???????? = = ()909090????Resolution range (?)40.4C1.9 (2.0C1.9)50.0C1.8 (1.9C1.8)50C1.8 (1.9C1.8)????The and ? electron densities, and therefore the phenyl group was put in a position that partially fitted to the electron densities. The 2 2? map of the phenyl ring was ambiguous even after refinement, and the ambiguity was the same in each monomer in the asymmetric unit. There was no significant change in the (/)8-fold and loops among the apo and two complex structures. Open in a separate window FIGURE 2. Tight binding of the disaccharide in the deep active site. and and indicate the bound PhPA (? omit map electron densities of PhPA and BsPA are in contoured at 3.0 . model, whereas the represents hydrogen bonds between amino acids and PhPA. Schematic diagram represents contacts and distance between BsPA Ginkgetin and amino acids. Electron densities of post-translationally modified glycans were found at two Ntransition state. Disaccharide Glycone Recognition in Subsite ?2 and Subsite ?1 Subsite ?2 held the -1,6-Xyl moiety by six amino acids, Glu-470, Ser-473, and Gln-477 with hydrogen bonds and Val-386, Phe-389, and Phe-479 with hydrophobic contacts (Fig. 2model. The and represent PhPA and BsPA, respectively. The represents a hydrogen bond between Tyr-209 and the succinimide moiety of BsPA. represents the -1,6-Xyl of the bound BsPA in the complex structure. The hydrogen bonds of equatorial 4-hydroxy of -1,6-Xyl moiety (is the axial 4-hydroxy of -1,6-l-Ara (is the 5-hydroxymethyl of -1,6-Glc (5-CH2OH) in -gentiobioside. The models indicate steric hindrance of 5-CH2OH by less distance than van der Waals radii to Phe-389. model. The DIMBOA -glucoside is shown by the value for -vicianoside being seven times greater than that for -primeveroside (16). The structural difference between -primeveroside and -vicianoside is the stereochemistry of the -1,6-linked sugars, 6-(DG) because aglycone binding of this enzyme is well studied in complexes with DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) -d-glucopyranoside (PDB code Ginkgetin 1E56) (34). The barrel-fold of PD was very similar to that of DG with an overall root mean square deviation of 1 1.06 ? in the superimposed structure. The subsite +1 of DG offers Phe-198, Phe-205, Trp-378, and Phe-466 as important residues for aglycone binding. These sites are also involved in aglycone binding of dhurrinase reported with high resolution constructions (19). The related amino acid residues of PD were investigated Ginkgetin in the crystal structure of subsite +1. Gly-210, Leu-217, Ala-387, and Leu-472 were recognized in PD, and corresponded to the aglycone-recognizing residues of DG, Phe-198, Phe-205, Trp-378, and Phe-466 (34), respectively (Fig. 3= 26 m) of BsPA filling subsite +1 with the large aglycone. The fact the hydrophobic part of -1,6-Xyl faces the aglycone suggests that subsites ?2 and +1 could bind substrate inside a concerted manner. The homology modeling of a diglycosdase cloned from expected the 6-are disaccharide-specific glycosidases, and the sequences are GH1 -glucosidases. indicate assorted residues in the substrate-binding site. The character shows highly conserved glucose binding residues in GH1 -glucosidase. The character shows conserved residues in the aligned sequences. The region 15C481 including substrate-binding site is definitely demonstrated in 507 amino acids of PD. Acknowledgment We.The corresponding amino acid residues of PD were investigated in the crystal structure of subsite +1. Gly-210, Leu-217, Ala-387, and Leu-472 were recognized in PD, and corresponded to the aglycone-recognizing residues of DG, Phe-198, Phe-205, Trp-378, and Phe-466 (34), respectively (Fig. of tea leaves raise the levels of these compounds to 10- to 40-collapse (15). Plucking, withering, and shaking methods during the manufacture of oolong tea wounds the fresh leaves to secrete volatile compounds as sweet, blossom, and honey aromatic flavors. During this process PD hydrolyzes -primeverosides and releases aromatic aglycones as volatile defensive compounds against stress. The hydrolytic activity of PD is definitely specific to -primeverosides, highly glycone-specific for 6-= 140 and 26 m, respectively (20). The monosaccharide glycoside analogue, -glucosylamidine, has no inhibitory activity at 500 m, consistent with the fact that PD barely hydrolyzes -d-glucopyranosides. Consequently, x-ray crystal analysis of PD in complex with (?)59.6, 88.8, 195.260.0, 88.2, 195.659.1, 89.9, 195.2???????? = = ()909090????Resolution range (?)40.4C1.9 (2.0C1.9)50.0C1.8 (1.9C1.8)50C1.8 (1.9C1.8)????The and ? electron densities, and therefore the phenyl group was put in a position that partially fitted to the electron densities. The 2 2? map of the phenyl ring was ambiguous actually after refinement, and the ambiguity was the same in each monomer in the asymmetric unit. There was no significant switch in the (/)8-collapse and loops among the apo and two complex structures. Open in a separate window Number 2. Tight binding of the disaccharide in the deep active site. and and indicate the bound PhPA (? omit map electron densities of PhPA and BsPA are in contoured at 3.0 . model, whereas the represents hydrogen bonds between amino acids and PhPA. Schematic diagram represents contacts and range between BsPA and amino acids. Electron densities of post-translationally revised glycans were found at two Ntransition state. Disaccharide Glycone Acknowledgement in Subsite ?2 and Subsite ?1 Subsite ?2 held the -1,6-Xyl moiety by six amino acids, Glu-470, Ser-473, and Gln-477 with hydrogen bonds and Val-386, Phe-389, and Phe-479 with hydrophobic contacts (Fig. 2model. The and represent PhPA and BsPA, respectively. The represents a hydrogen relationship between Tyr-209 and the succinimide moiety of BsPA. represents the -1,6-Xyl of the bound BsPA in the complex structure. The hydrogen bonds of equatorial 4-hydroxy of -1,6-Xyl moiety (is the axial 4-hydroxy of -1,6-l-Ara (is the 5-hydroxymethyl of -1,6-Glc (5-CH2OH) in -gentiobioside. The models indicate steric hindrance of 5-CH2OH by less distance than vehicle der Waals radii to Phe-389. model. The DIMBOA -glucoside is definitely shown by the value for -vicianoside becoming seven times greater than that for -primeveroside (16). The structural difference between -primeveroside and -vicianoside is the stereochemistry of the -1,6-linked sugars, 6-(DG) because aglycone binding of this enzyme is definitely well analyzed in complexes with DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) -d-glucopyranoside (PDB code 1E56) (34). The barrel-fold of PD was very similar to that of DG with an overall root mean square deviation of 1 1.06 ? in the superimposed structure. The subsite +1 of DG offers Phe-198, Phe-205, Trp-378, and Phe-466 as important residues for aglycone binding. These sites are also involved in aglycone binding of dhurrinase reported with high resolution constructions (19). The related amino acid residues of PD were investigated in the crystal structure of subsite +1. Gly-210, Leu-217, Ala-387, and Leu-472 were recognized in PD, and corresponded to the aglycone-recognizing residues of DG, Phe-198, Phe-205, Trp-378, and Phe-466 (34), respectively (Fig. 3= 26 m) of BsPA filling subsite +1 with the large aglycone. The fact the hydrophobic part of -1,6-Xyl faces the aglycone suggests that subsites ?2 and +1 could bind substrate inside a concerted manner. The homology modeling of a diglycosdase cloned from expected the 6-are disaccharide-specific glycosidases, and the sequences are GH1 -glucosidases. indicate assorted residues in the substrate-binding site. The character indicates highly conserved glucose binding residues in GH1 -glucosidase. The character shows conserved residues in the aligned sequences. The region 15C481 including substrate-binding site is definitely demonstrated in 507 amino acids of PD. Acknowledgment We say thanks to Prof. Masashi Miyano for essential reading Ginkgetin of the manuscript and help with number preparation. *This work was supported KPSH1 antibody in part by Ministry of Education, Tradition, Sports, Technology, and Technology of Japan Grants-in-Aids 13460049 and 16380079 and the MEXT (Ministry of Education, Tradition, Sports, Science and Technology)-supported Program for the Strategic Research Foundation at Private Universities (2013C2017). The atomic coordinates and structure factors (codes 3WQ4, 3WQ5, and 3WQ6) have been deposited in the Protein Data Lender (http://wwpdb.org/). 2The abbreviations used are: PD-primeverosidaseFHfurcatin hydrolaseVHvicianin hydrolaseDG(2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one)-glucoside hydrolaseXyld-xylopyranosylGlcd-glucopyranosylAral-arabinopyranosylGH1glycoside hydrolase family 1DIMBOA2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-onePhPA2-phenyl-efficacy of herb volatiles for inhibiting the growth of fruit and vegetable decay microorganisms. J. Agric. Food Chem. 50, 6371C6377 [PubMed] [Google Scholar] 12. Griffin S. G., Wyllie S. G.,.(2013) Enzymatic and structural characterization of hydrolysis of gibberellin A4 glucosyl ester by a rice -d-glucosidase. PD hydrolyzes -primeverosides and releases aromatic aglycones as volatile defensive compounds against stress. The hydrolytic activity of PD is usually specific to -primeverosides, highly glycone-specific for 6-= 140 and 26 m, respectively (20). The monosaccharide glycoside analogue, -glucosylamidine, has no inhibitory activity at 500 m, consistent with the fact that PD barely hydrolyzes -d-glucopyranosides. Therefore, x-ray crystal analysis of PD in complex with (?)59.6, 88.8, 195.260.0, 88.2, 195.659.1, 89.9, 195.2???????? = = ()909090????Resolution range (?)40.4C1.9 (2.0C1.9)50.0C1.8 (1.9C1.8)50C1.8 (1.9C1.8)????The and ? electron densities, and therefore the phenyl group was put in a position that partially fitted to the electron densities. The 2 2? map of the phenyl ring was ambiguous even after refinement, and the ambiguity was the same in each monomer in the asymmetric unit. There was no significant switch in the (/)8-fold and loops among the apo and two complex structures. Open in a separate window Physique 2. Tight binding of the disaccharide in the deep active site. and and indicate the bound PhPA (? omit map electron densities of PhPA and BsPA are in contoured at 3.0 . model, whereas the represents hydrogen bonds between amino acids and PhPA. Schematic diagram represents contacts and distance between BsPA and amino acids. Electron densities of post-translationally altered glycans were found at two Ntransition state. Disaccharide Glycone Acknowledgement in Subsite ?2 and Subsite ?1 Subsite ?2 held the -1,6-Xyl moiety by six amino acids, Glu-470, Ser-473, and Gln-477 with hydrogen bonds and Val-386, Phe-389, and Phe-479 with hydrophobic contacts (Fig. 2model. The and represent PhPA and BsPA, respectively. The represents a hydrogen bond between Tyr-209 and the succinimide moiety of BsPA. represents the -1,6-Xyl of the bound BsPA in the complex structure. The hydrogen bonds of equatorial 4-hydroxy of -1,6-Xyl moiety (is the axial 4-hydroxy of -1,6-l-Ara (is the 5-hydroxymethyl of -1,6-Glc (5-CH2OH) in -gentiobioside. The models indicate steric hindrance of 5-CH2OH by less distance than van der Waals radii to Phe-389. model. The DIMBOA -glucoside is usually shown by the value for -vicianoside being seven times greater than that for -primeveroside (16). The structural difference between -primeveroside and -vicianoside is the stereochemistry of the -1,6-linked sugars, 6-(DG) because aglycone binding of this enzyme is usually well analyzed in complexes with DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) -d-glucopyranoside (PDB code 1E56) (34). The barrel-fold of PD was very similar to that of DG with an overall root mean square deviation of 1 1.06 ? in the superimposed structure. The subsite +1 of DG has Phe-198, Phe-205, Trp-378, and Phe-466 as crucial residues for aglycone binding. These sites are also involved in aglycone binding of dhurrinase reported with high resolution structures (19). The corresponding amino acid residues of PD were investigated in the crystal structure of subsite +1. Gly-210, Leu-217, Ala-387, and Leu-472 were recognized in PD, and corresponded to the aglycone-recognizing residues of DG, Phe-198, Phe-205, Trp-378, and Phe-466 (34), respectively (Fig. 3= 26 m) of BsPA filling subsite +1 with the large aglycone. The fact that this hydrophobic side of -1,6-Xyl faces the aglycone suggests that subsites ?2 and +1 could bind substrate in a concerted manner. The homology modeling of a diglycosdase cloned from expected that this 6-are disaccharide-specific glycosidases, and the sequences are GH1 Ginkgetin -glucosidases. indicate varied residues in the substrate-binding site. The character indicates highly conserved glucose binding residues in GH1 -glucosidase. The character indicates conserved residues in the aligned sequences. The region 15C481 including substrate-binding site is usually shown in 507 amino acids of PD. Acknowledgment We thank Prof. Masashi Miyano for crucial reading of the manuscript and help with physique preparation. *This work was supported in part by Ministry of Education, Culture, Sports, Science, and Technology of Japan Grants-in-Aids 13460049 and 16380079 and the MEXT (Ministry of Education, Culture, Sports, Science and Technology)-supported Program for the Strategic Research Foundation at Private Universities (2013C2017). The atomic coordinates and structure factors (codes 3WQ4, 3WQ5, and 3WQ6) have been deposited in the Protein Data Lender (http://wwpdb.org/). 2The abbreviations used are: PD-primeverosidaseFHfurcatin hydrolaseVHvicianin.(2006) Activation of glucosidase via stress-induced polymerization rapidly increases active pools of abscisic acid. compounds to 10- to 40-fold (15). Plucking, withering, and shaking procedures during the manufacture of oolong tea wounds the fresh leaves to secrete volatile compounds as sweet, blossom, and honey aromatic flavors. During this process PD hydrolyzes -primeverosides and releases aromatic aglycones as volatile defensive compounds against stress. The hydrolytic activity of PD is usually specific to -primeverosides, highly glycone-specific for 6-= 140 and 26 m, respectively (20). The monosaccharide glycoside analogue, -glucosylamidine, has no inhibitory activity at 500 m, consistent with the fact that PD barely hydrolyzes -d-glucopyranosides. Therefore, x-ray crystal analysis of PD in complex with (?)59.6, 88.8, 195.260.0, 88.2, 195.659.1, 89.9, 195.2???????? = = ()909090????Resolution range (?)40.4C1.9 (2.0C1.9)50.0C1.8 (1.9C1.8)50C1.8 (1.9C1.8)????The and ? electron densities, and therefore the phenyl group was put in a position that partially fitted to the electron densities. The 2 2? map of the phenyl ring was ambiguous even after refinement, and the ambiguity was the same in each monomer in the asymmetric unit. There was no significant switch in the (/)8-fold and loops among the apo and two complex structures. Open in a separate window Physique 2. Tight binding of the disaccharide in the deep active site. and and indicate the bound PhPA (? omit map electron densities of PhPA and BsPA are in contoured at 3.0 . model, whereas the represents hydrogen bonds between amino acids and PhPA. Schematic diagram represents contacts and distance between BsPA and amino acids. Electron densities of post-translationally altered glycans were found at two Ntransition state. Disaccharide Glycone Acknowledgement in Subsite ?2 and Subsite ?1 Subsite ?2 held the -1,6-Xyl moiety by six amino acids, Glu-470, Ser-473, and Gln-477 with hydrogen bonds and Val-386, Phe-389, and Phe-479 with hydrophobic contacts (Fig. 2model. The and represent PhPA and BsPA, respectively. The represents a hydrogen bond between Tyr-209 and the succinimide moiety of BsPA. represents the -1,6-Xyl of the bound BsPA in the complex structure. The hydrogen bonds of equatorial 4-hydroxy of -1,6-Xyl moiety (may be the axial 4-hydroxy of -1,6-l-Ara (may be the 5-hydroxymethyl of -1,6-Glc (5-CH2OH) in -gentiobioside. The versions indicate steric hindrance of 5-CH2OH by much less distance than vehicle der Waals radii to Phe-389. model. The DIMBOA -glucoside can be shown by the worthiness for -vicianoside becoming seven times higher than that for -primeveroside (16). The structural difference between -primeveroside and -vicianoside may be the stereochemistry from the -1,6-connected sugar, 6-(DG) because aglycone binding of the enzyme can be well researched in complexes with DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) -d-glucopyranoside (PDB code 1E56) (34). The barrel-fold of PD was nearly the same as that of DG with a standard root mean rectangular deviation of just one 1.06 ? in the superimposed framework. The subsite +1 of DG offers Phe-198, Phe-205, Trp-378, and Phe-466 as important residues for aglycone binding. These websites are also involved with aglycone binding of dhurrinase reported with high res constructions (19). The related amino acidity residues of PD had been looked into in the crystal framework of subsite +1. Gly-210, Leu-217, Ala-387, and Leu-472 had been determined in PD, and corresponded towards the aglycone-recognizing residues of DG, Phe-198, Phe-205, Trp-378, and Phe-466 (34), respectively (Fig. 3= 26 m) of BsPA filling up subsite +1 using the huge aglycone. The actual fact how the hydrophobic part of -1,6-Xyl encounters the aglycone shows that subsites ?2 and +1 could bind substrate inside a concerted way. The homology modeling of the diglycosdase cloned from anticipated how the 6-are disaccharide-specific glycosidases, as well as the sequences are GH1 -glucosidases. indicate assorted residues in the substrate-binding site. The type indicates extremely conserved glucose binding residues in GH1 -glucosidase. The type shows conserved residues in the aligned sequences. The spot 15C481 including substrate-binding site.