Knowledge‐based protein modelling and design

Blundell, Tom L.; Carney, Devon; Gardner, Stephen P.; Hayes, Fiona; Howlin, Brendan J.; Hubbard, Tim; Overington, John P.; Singh, Diljeet Athwal; Sibanda, B. L.; Sutcliffe, Michael J.

doi:10.1111/j.1432-1033.1988.tb13917.x

Cited by 244 publications

(117 citation statements)

References 40 publications

(28 reference statements)

Supporting

Mentioning

116

Contrasting

Unclassified

Order By: Relevance

“…From this preliminary model, a number of key interactions were identified, and these were restrained during refinement (Blundell et al, 1988). Finally, an exhaustive search of the possible orientations of the G2OG and G6OG moieties in the active site was undertaken, using information on the Ϫ1 subsite of the G4SG moiety of the structure of the S-cellobioside/enzyme complex.…”

Section: Molecular Modeling Of ␤-D-glucopyranosyl-(1→2)-d-glucose Andmentioning

confidence: 99%

“…The molecular models of the barley ␤-D-glucan glucohydrolase with bound ␤-D-glucopyranosyl-(1→2)-D-glucose (sophorose or G2OG) and ␤-D-glucopyranosyl-(1→6)-D-glucose (gentiobiose or G6OG) were constructed manually with the program SYBYL 6.62 (Blundell et al, 1988) and involved two stages. The first stage represented energy minimization of the ␤-D-glucan glucohydrolase structure ) on amino acid residues within a 15-Å radius of the active site.…”

Section: Molecular Modeling Of ␤-D-glucopyranosyl-(1→2)-d-glucose Andmentioning

confidence: 99%

See 1 more Smart Citation

Structural Basis for Broad Substrate Specificity in Higher Plant β-d-Glucan Glucohydrolases

et al. 2002

View full text Add to dashboard Cite

Family 3 ␤ -D -glucan glucohydrolases are distributed widely in higher plants. The enzymes catalyze the hydrolytic removal of ␤ -D -glucosyl residues from nonreducing termini of a range of ␤ -D -glucans and ␤ -D -oligoglucosides. Their broad specificity can be explained by x-ray crystallographic data obtained from a barley ␤ -D -glucan glucohydrolase in complex with nonhydrolyzable S -glycoside substrate analogs and by molecular modeling of enzyme/substrate complexes. The glucosyl residue that occupies binding subsite ؊ 1 is locked tightly into a fixed position through extensive hydrogen bonding with six amino acid residues near the bottom of an active site pocket. In contrast, the glucosyl residue at subsite ؉ 1 is located between two Trp residues at the entrance of the pocket, where it is constrained less tightly. The relative flexibility of binding at subsite ؉ 1, coupled with the projection of the remainder of bound substrate away from the enzyme's surface, means that the overall active site can accommodate a range of substrates with variable spatial dispositions of adjacent ␤ -D -glucosyl residues. The broad specificity for glycosidic linkage type enables the enzyme to perform diverse functions during plant development. INTRODUCTION␤ -D -Glucan glucohydrolases have been purified and characterized from barley seedlings, maize coleoptiles, soybean cultures, Acacia cells, nasturtium cells, and cultured tobacco cells (Cline and Albersheim, 1981;Nari et al., 1982;Lienart et al., 1986;Labrador and Nevins, 1989;Hrmova et al., 1996;Kotake et al., 1997;Crombie et al., 1998;Kim et al., 2000;Koizumi et al., 2000). They can hydrolyze glycosidic linkages in several ␤ -D -glucans, in ␤ -D -oligoglucosides containing (1 → 2)-, (1 → 3)-, (1 → 4)-, or (1 → 6)-linkages, in aryl ␤ -D -glucosides such as 4 Ј -nitrophenyl ␤ -D -glucopyranoside (4NPGlc), and in some ␤ -D -oligoxyloglucosides (Crombie et al., 1998;Hrmova and Fincher, 1998;Kim et al., 2000). The barley ␤ -D -glucan glucohydrolases also hydrolyze cyanogenic ␤ -D -glucosides, albeit with low activity (M. Hrmova and G.B. Fincher, unpublished data). Single Glc molecules are released from the nonreducing termini of these substrates, with retention of the anomeric configuration (Hrmova et al., 1996). Their broad substrate specificity makes it difficult to assign these higher plant ␤ -D -glucan glucohydrolases to current Enzyme Commission classes; therefore, they have been described variously as ␤ -D -glucan glucohydrolases, (1 → 3)-␤ -D -glucan exohydrolases, and ␤ -D -glucosidases. Nevertheless, they can be classified according to the structural criteria of Henrissat (1991) and fall into the family 3 group of glycoside hydrolases (http://afmb.cnrsmrs.fr/CAZY/).The distribution of the broad-specificity ␤ -D -glucan glucohydrolases in various tissues of monocotyledons and dicotyledons, together with the presence of expressed sequence tags in gymnosperm sequence databases (e.g., Pinus taeda ), suggests that they may play a fundamental role in plant growth and development...

show abstract

Section: Molecular Modeling Of ␤-D-glucopyranosyl-(1→2)-d-glucose Andmentioning

confidence: 99%

Section: Molecular Modeling Of ␤-D-glucopyranosyl-(1→2)-d-glucose Andmentioning

confidence: 99%

Structural Basis for Broad Substrate Specificity in Higher Plant β-d-Glucan Glucohydrolases

et al. 2002

View full text Add to dashboard Cite

show abstract

“…Regions between two structurally conserved regions were defined as loops. UGT85B1 models were built with the homology-modeling program COMPOSER (Blundell et al, 1988) of the Sybyl software (SYBYL). High-resolution crystal structures of GtfA and GtfB were employed in building structurally conserved regions of the UGT85B1 model.…”

Section: Comparative Modeling Of Ugt85b1mentioning

confidence: 99%

Determination of Catalytic Key Amino Acids and UDP Sugar Donor Specificity of the Cyanohydrin Glycosyltransferase UGT85B1 from Sorghum bicolor. Molecular Modeling Substantiated by Site-Specific Mutagenesis and Biochemical Analyses

Thorsøe¹,

Bak²,

Olsen³

et al. 2005

Plant Physiology

View full text Add to dashboard Cite

Plants produce a plethora of structurally diverse natural products. The final step in their biosynthesis is often a glycosylation step catalyzed by a family 1 glycosyltransferase (GT). In biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor, the UDPglucosyltransferase UGT85B1 catalyzes the conversion of p-hydroxymandelonitrile into dhurrin. A structural model of UGT85B1 was built based on hydrophobic cluster analysis and the crystal structures of two bacterial GTs, GtfA and GtfB, which each showed approximately 15% overall amino acid sequence identity to UGT85B1. The model enabled predictions about amino acid residues important for catalysis and sugar donor specificity. p-Hydroxymandelonitrile and UDP-glucose (Glc) were predicted to be positioned within hydrogen-bonding distance to a glutamic acid residue in position 410 facilitating sugar transfer. The acceptor was packed within van der Waals distance to histidine H23. Serine S391 and arginine R201 form hydrogen bonds to the pyrophosphate part of UDP-Glc and hence stabilize binding of the sugar donor. Docking of UDP sugars predicted that UDP-Glc would serve as the sole donor sugar in UGT85B1. This was substantiated by biochemical analyses. The predictive power of the model was validated by site-directed mutagenesis of selected residues and using enzyme assays. The modeling approach has provided a tool to design GTs with new desired substrate specificities for use in biotechnological applications. The modeling identified a hypervariable loop (amino acid residues 156-188) that contained a hydrophobic patch. The involvement of this loop in mediating binding of UGT85B1 to cytochromes P450, CYP79A1, and CYP71E1 within a dhurrin metabolon is discussed.A glycosyltransferase (GT) is an enzyme that attaches a sugar molecule to a specific acceptor and thereby creates a glycosidic bond. GTs are found in all phylae. The nature of the acceptor molecules used is highly diverse, whereas the donor molecules typically are restricted to monosaccharides with either D-or L-configuration linked to a nucleotide (UDP/GDP/ CMP/(d)TDP; Leloir, 1964). Glycosylation reactions constitute an integral part of both primary and secondary metabolism. The final step in plant natural product synthesis is often a glycosylation reaction that serves to stabilize, solubilize, and provide a safe storage form of potentially toxic aglycones. In Sorghum bicolor, the GT UGT85B1 catalyzes glucosylation of Tyr-derived p-hydroxymandelonitrile to yield the cyanogenic glucoside dhurrin (Jones et al., 1999). In planta, neither p-hydroxymandelonitrile nor its degradation products are detectable, indicating that the glucosylation reaction is highly efficient and channeled (Møller and Conn, 1980). This observation has been substantiated by metabolome and transcriptome analyses of transgenic Arabidopsis (Arabidopsis thaliana) plants expressing the entire dhurrin pathway (Tattersall et al., 2001;Jørgensen et al., 2005;Kristensen et al., 2005), as well as by confocal laser-scanning microscopy studies ...

show abstract

“…The homology modeling COMPOSER program (Blundell et al, 1988) of the Sybyl software (Tripos Associates, St. Louis) was used to build the structurally conserved regions of the lectin model. Loops were built from a library containing all legume lectins structures, and their geometries were optimized.…”

Section: Molecular Modelingmentioning

confidence: 99%

Characterization of Four Lectin-Like Receptor Kinases Expressed in Roots of Medicago truncatula. Structure, Location, Regulation of Expression, and Potential Role in the Symbiosis with Sinorhizobium meliloti

et al. 2003

View full text Add to dashboard Cite

To study the role of LecRK (lectin-like receptor kinase) genes in the legume-rhizobia symbiosis, we have characterized the four Medicago truncatula Gaernt. LecRK genes that are most highly expressed in roots. Three of these genes, MtLecRK7;1, MtLecRK7;2, and MtLecRK7;3, encode proteins most closely related to the Class A LecRKs of Arabidopsis, whereas the protein encoded by the fourth gene, MtLecRK1;1, is most similar to a Class B Arabidopsis LecRK. All four genes show a strongly enhanced root expression, and detailed studies on MtLecRK1;1 and MtLecRK7;2 revealed that the levels of their mRNAs are increased by nitrogen starvation and transiently repressed after either rhizobial inoculation or addition of lipochitooligosaccharidic Nod factors. Studies of the MtLecRK1;1 and MtLecRK7;2 proteins, using green fluorescent protein fusions in transgenic M. truncatula roots, revealed that they are located in the plasma membrane and that their central transmembrane-spanning helix is required for correct sorting. Moreover, their lectin-like domains appear to be highly glycosylated. Of the four proteins, only MtLecRK1;1 shows a high conservation of key residues implicated in monosaccharide binding, and molecular modeling revealed that this protein may be capable of interacting with Nod factors. However, no increase in Nod factor binding was found in roots overexpressing a fusion in which the kinase domain of this protein had been replaced with green fluorescent protein. Roots expressing this fusion protein however showed an increase in nodule number, suggesting that expression of MtLecRK1;1 influences nodulation. The potential role of LecRKs in the legumerhizobia symbiosis is discussed.The lectin-like receptor kinases (LecRKs) are a class of proteins originally described from Arabidopsis (Hervé et al., 1996). They have a structure similar to other plant receptor-like kinases (RLKs; Shiu and Bleecker, 2001;Cock et al., 2002) with an N-terminal targeting signal, a presumably extracellular domain, a single transmembrane (TM)-spanning helix, and a cytosolic kinase domain. The Arabidopsis genome contains over 610 RLKs that have been shown to be monophylogenetic with respect to the kinase domain and most closely related to the fruitfly (Drosophila melanogaster) Pelle and related animal cytoplasmic kinases (Shiu and Bleecker, 2001). In cases in which they have been tested, these kinases have been shown to have specificity for phosphorylation on Ser/Thr residues (Shiu and Bleecker, 2001).The plant RLKs can be grouped into more than 21 structural classes based on their extracellular domains (Shiu and Bleecker, 2001). The LecRKs represent one such class in which the extracellular domain is related to legume lectins. The Arabidopsis genome contains at least 42 LecRK sequences that have been grouped into three classes (A-C) plus an additional nine sequences of related soluble lectins (Barre et al., 2002). Searches in plant databases reveal that LecRK genes are widespread in higher plants, but apart from Arabidopsis (Hervé et al., 1996,...

show abstract

Knowledge‐based protein modelling and design

Cited by 244 publications

References 40 publications

Structural Basis for Broad Substrate Specificity in Higher Plant β-d-Glucan Glucohydrolases

Structural Basis for Broad Substrate Specificity in Higher Plant β-d-Glucan Glucohydrolases

Determination of Catalytic Key Amino Acids and UDP Sugar Donor Specificity of the Cyanohydrin Glycosyltransferase UGT85B1 from Sorghum bicolor. Molecular Modeling Substantiated by Site-Specific Mutagenesis and Biochemical Analyses

Characterization of Four Lectin-Like Receptor Kinases Expressed in Roots of Medicago truncatula. Structure, Location, Regulation of Expression, and Potential Role in the Symbiosis with Sinorhizobium meliloti

Contact Info

Product

Resources

About