Mutation of UDP-glucose binding motif residues lead to increased affinity for ADP-glucose in sugarcane sucrose phosphate synthase

Kurniah, Nuriyah Inda and Sawitri, Widhi Dyah and Rohman, Muhammad Saifur and Nugraha, Yudhi and Hase, Toshiharu and Sugiharto, Bambang (2021) Mutation of UDP-glucose binding motif residues lead to increased affinity for ADP-glucose in sugarcane sucrose phosphate synthase. MOLECULAR BIOLOGY REPORTS, 48 (2). pp. 1697-1706. ISSN 0301-4851

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Abstract

Plant sucrose-phosphate synthase (SPS) contains a glycosyltransferase domain, which specifically catalyzes reactions with the nucleotide sugar uridine diphosphate glucose (UDP-G) as a donor substrate. Unlike plant SPS, bacterial SPS is predicted to bind other nucleotide sugars, such as adenosine diphosphate glucose (ADP-G). This study aimed to identify the UDP-G binding site of sugarcane (Saccharum officinarum) SPS (SoSPS1) and to improve its affinity for ADP-G by site-directed mutagenesis. To achieve targeted mutagenesis, amino acid distribution and comparative modeling studies were performed, followed by site-directed mutagenesis of SoSPS1 in the putative UDP-G binding motif. The N-terminal deletion of SoSPS1 ( increment N-SoSPS1) was used for enzymatic analysis. The results showed that mutations in the R-X-4-K, E-X-7-E, and H-X-5-V motifs significantly affect UDP-G and ADP-G binding. Mutations at R496 and K501 severely attenuate the affinity for UDP-G. Additionally, alanine substitutions at E591 and V570 decreased the UDP-G affinity but remarkably increased its ADP-G affinity. The R-X-4-K motif plays a crucial role in the UDP-G binding site and catalytic activity of plant SPS; thus, its alteration to other amino acids was not viable. The E-X-7-E and H-X-5-V motifs may bind to the nucleotide glucose substrate, indicating that these motifs are involved in substrate specificity. These results agree with substrate docking simulations at the mutated residue positions, supporting the experimental results. These results demonstrate that mutation of E591 and V570 severely attenuated the UDP-G affinity, while retaining its activity against ADP-G, offering strategic insights into increasing sucrose synthesis and plant growth.

Item Type: Article
Uncontrolled Keywords: Sucrose phosphate synthase; Adenine diphosphate glucose; Uridine diphosphate glucose; Glycosyltransferase; Site-directed mutagenesis
Subjects: S Agriculture > S Agriculture (General)
Divisions: Faculty of Agriculture > Department of Agronomy
Depositing User: Sri JUNANDI
Date Deposited: 16 Oct 2024 01:58
Last Modified: 16 Oct 2024 01:58
URI: https://ir.lib.ugm.ac.id/id/eprint/9322

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