Identification of UDP-glucose binding site in glycosyltransferase domain of sucrose phosphate synthase from sugarcane (Saccharum officinarum) by structure-based site-directed mutagenesis

Abstract

Sucrose phosphate synthase (SPS) is believed to be the key enzyme for controlling the biosynthesis of sucrose. SPSs consist of a functional glycosyltransferase domain that shares conserved residues with the glycosyltransferase domain of sucrose biosynthesis-related protein. The formation of sucrose-6-phosphate is catalyzed by SPS with the transfer of a glycosyl group of uridine diphosphate glucose (UDP-G) as an activated donor sugar to a fructose-6-phosphate as a sugar acceptor. However, understanding of the mechanism of catalytic and substrate binding in SPS is very limited. Based on amino acid sequence alignments with several enzymes that belong to the glycosyltransferase family, the UDP-G binding sites that might be critical for catalytic mechanism were identified. Here, we report that single point mutation of R496, D498, and V570 located in the proposed UDP-G binding site led to less active or complete loss of enzyme activity. Through structure-based site-directed mutagenesis and biochemical studies, the results indicated that these residues contribute to the catalytic activity of plant SPS. Moreover, understanding of the UDP-G binding site provides an insight into new strategies for enzyme engineering and redesigning a catalytic mechanism for UDP.

Keywords: Glycosyltransferase; Site-directed mutagenesis; Sucrose phosphate synthase; Sugarcane; Uridine diphosphate glucose.

Fig. 1

Comparison of residues in glycosyltransferases group. a Multiple sequence alignment of SPS in sugarcane (SoSPS1), SPS in non-photosynthetic bacterial H. orenii (HoSPS), sucrose synthase in A. thaliana (AtSuSy), sucrose synthase in bacteria N. europaea (NeSuSy), and glycogen synthase from E. coli (EcGS). The three highly conserved and critical residues that consist of arginine, lysine, and glutamic acid are shown in red. The residues that are predicted to be involved in nucleotide sugar interaction is shown in blue. b The UDP binding site residues at positions corresponding to Arg-580, Asp-582, Lys-585, and Glu-675 for diphosphate moiety binding and Gln-648 and Asn-654 for uridine moiety binding of A. thaliana sucrose synthase (PDB ID: 3S28). c Predicted interaction hydrogen bonding between Ser-269 and Arg-270 in bacterial SPS (PDB ID: 2R60)

Fig. 2

Site-directed mutagenesis and expression of recombinant SoSPS1 in E. coli . a The resulting SPS mutation at glycosyltransferase domain was transformed into E. coli overnight and the total cell proteins were detected by western blotting using antibodies against polypeptide of SPS. b The affinity purification of His-tagged ∆N-SoSPS1 as monitored by SDS-PAGE and Coomassie blue staining. Lane 1 The eluted sample from DE52 anion exchange cellulose. Lanes 2–5 Washing steps of affinity His-tag purification with increasing concentration of imidazole 0 and 20 mM. Lanes 6–10 The fraction of samples eluted successively with the buffer containing 100 mM imidazole. Lanes 11–12 The purified SPSs were concentrated and exchanged to buffer composed of 50 mM Tris-HCl, pH 7.5 and 150 mM NaCl. Lane 13: Protein molecular marker (Biorad-Dual Color Standards). c Activity of SPS mutants (S495A, R496A, P497A, and D498A) compared to wild-type ∆N-SoSPS1 and vector pTrcHis as a control. Arrows represent the targeted proteins