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 UDP-glucosyltransferase 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.

Figure 1.

Structural amino acid sequence alignment of the S. bicolor GT UGT85B1 with the A. orientalis GTs GtfA and GtfB. Secondary structure elements are as follows: β-strands (turquoise coloring) and α-helices (purple coloring). Loops A, B, C, and D, which were removed in the UGT85B1 model, are shown with red bars. Amino acid residues further analyzed by mutagenesis are shown with a green background. Amino acid residues in red italics were not seen in the GtfB crystal structure, indicating conformational flexibility for these regions. The hydrophobic patch in loop B is shown with a yellow background. The PSPG motif is underlined. α-Helix 7 links the N- and C-terminal domains.

Figure 2.

Schematic presentation of the overall architecture of the GtfA crystal structure (A) and the UGT85B1 model (B). α-Helices are shown in purple and β-sheets in turquoise. N and C termini are indicated. Substrates are shown as ball-and-stick presentations in the catalytic pocket of both enzymes with GtfA, including both the TDP part of the donor molecule TDP-epi-vancosamine and the acceptor molecule vancomycin, and UGT85B1 modeled with the donor molecule UDP-Glc and the acceptor p-hydroxymandelonitrile. Green star, position of removed loop A; yellow stars, beginning and ending of removed loop B; purple stars, beginning and ending of removed loop C; blue star, position of removed loop D.

Figure 3.

Multiple alignment of plant family 1 GTs. Only the region between α-helices 4 and 5 showing the hydrophobic cluster, loops B and C (red boxes), as well as β-strand 5 is shown. The UGT numbers correspond to the nomenclature at http://www.p450.kvl.dk//Arab_ugts/table.shtml. R201 is encircled.

Figure 4.

Predicted interactions in the catalytic pocket of UGT85B1 with the donor molecule UDP-Glc and the acceptor molecules p-hydroxymandelonitrile (A) and geraniol (B). Substrates are indicated by bold ball-and-stick presentations. Hydrogen bonds between individual amino acid residues (in gray) and substrates are indicated by dashed green lines.

Figure 5.

Structure of UDP-Glc, UDP-Gal, UDP-GlcUA, and UDP-Xyl.

Figure 6.

Specificity of UGT85B1 toward different radiolabeled UDP sugars as monitored by TLC. A, UDP-Glc; B, UDP-GlcUA; C, UDP-Gal; D, UDP-Xyl. The arrow shows origin of application.

Figure 7.

The ability of UGT85B1 to catalyze mandelonitrile-glucoside formation as verified by LC-MS and LC-MS/MS. A, Total ion trace of a reaction mixture containing UGT85B1, UDP-Glc, and mandelonitrile and either UDP-Glc (solid line) or UDP-Gal (dashed line). The compound eluting at Rt = 11.6 min is prunasin and the compound eluting at Rt = 12.3 min is sambunigrin. B, Extracted ion monitoring (m/z 318) of a reaction mixture containing UGT85B1, UDP-Glc, and mandelonitrile. C, Fragmentation of the m/z ion 318 (♦) corresponding to the mandelonitrile-glucoside-Na⁺ adduct. m/z 290.9 corresponds to mandelonitrile-glucoside lacking hydrogen cyanide. m/z 184.9 corresponds to the sugar moiety.