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

Abstract

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 ...

Catalytic Key Amino Acids and UDP-Sugar Donor Specificity of a Plant Glucuronosyltransferase, UGT94B1: Molecular Modeling Substantiated by Site-Specific Mutagenesis and Biochemical Analyses

“…Residues in the PSPG motif of plant UGTs have previously been shown to be involved in sugar donor binding of UGTs (Kubo et al, 2004;Shao et al, 2005;Thorsoe et al, 2005;Offen et al, 2006). These residues are highly conserved and might not explain the various sugar specificities observed for plant UGTs.…”

“…Obtaining GT crystals has proven difficult and structure solution progresses slowly in comparison to the rate of appearance of new UGT sequences. To circumvent this bottleneck in the study of UGTs, homology modeling has proved to be a strong tool for the identification of potentially important residues (Hans et al, 2004;Thorsoe et al, 2005). The recently solved crystal structures of the first plant UGTs, MtUGT71G1 from M. truncatula (Shao et al, 2005) and VvGT1 from V. vinifera (Offen et al, 2006), essentially add confidence to the structures derived from homology modeling.…”

“…In plant UGTs, the most common sugar donor is UDP-Glc. Several conserved residues, most of which are found in the PSPG motif of plant UGTs, interact with the sugar donor (Shao et al, 2005;Thorsoe et al, 2005;Offen et al, 2006;Li et al, 2007). The sugar donor preference of a specific UGT is often very narrow, showing little or no activity with alternative sugars.…”

A single amino acid in the PSPG‐box plays an important role in the catalytic function of CaUGT2 (Curcumin glucosyltransferase), a Group D Family 1 glucosyltransferase from Catharanthus roseus

“…Investigation of the 3D-structures of betanidin 5-O-glucosyltransferase (B5GT) from Dorotheanthus bellidiformis [17] and cyanohydrin glucosyltransferase from Sorghum bicolor [18] by homology modeling, and of isoflavonoid 3 0 -O-glucosyltransferase from Medicago truncatula [19,20] and flavonoid 3-O-glucosyltransferase from Vitis vinifera [21] by X-ray crystallography, revealed the role of specific conserved amino acid residues in the PSPG-box that constitute the donor-sugar binding pockets. However, the roles of less well conserved amino acids within the motif that may determine the characteristics unique to particular enzymes such as substrate recognition and catalytic potential have been less closely examined.…”

Homology Modeling and Site-Directed Mutagenesis Reveal Catalytic Key Amino Acids of 3β-Hydroxysteroid-Dehydrogenase/C4-Decarboxylase from Arabidopsis

“…Homology modeling has been recently used to identify key amino acids in a number of plant enzymes (Thorsøe et al, 2005;Geissler et al, 2007;Osmani et al, 2008). In this work, a homology model of the tertiary structure of the monomeric 3bHSD/D was built and constitutes a novel example of the application of this methodology to a plant membrane-bound enzyme.…”