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.
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This work was supported by Ministry of Research, Technology and Higher Education of the Republic of Indonesia (Penelitian Unggulan Strategis Nasional and Penelitian Dasar Unggulan Perguruan Tinggi) and by the International Collaborative Research Program of Institute for Protein Research, Osaka University, ICR-14-03 and 05, Japan.
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Conflict of interest
Widhi Dyah Sawitri declares that she has no conflicts of interest. Siti Nurul Afidah declares that she has no conflicts of interest. Atsushi Nakagawa declares that he has no conflicts of interest. Toshiharu Hase declares that he has no conflicts of interest. Bambang Sugiharto declares that he has no conflicts of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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