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

摘要

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 residueswith the glycosyltransferase domain of sucrose biosynthesisrelated 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, theUDP-G binding sites thatmight be critical for catalytic mechanismwere 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.