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

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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 (∆N-SoSPS1) was used for enzymatic analysis. The results showed that mutations in the R-X4-K, E-X7-E, and H-X5-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-X4-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-X7-E and H-X5-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.

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Acknowledgements

This research was supported by the Indonesian Ministry of Education and Culture through a research Grant for Master Thesis Grant Number 975/UN25.3.1/LT/2020.

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Authors and Affiliations

  1. Laboratory of Molecular Biology and Biotechnology, Center for Development of Advanced Science and Technology (CDAST), University of Jember, Jl. Kalimantan No 37, Kampus Tegalboto, Jember, 68121, Indonesia

    Nuriyah Inda Kurniah & Bambang Sugiharto

  2. Department of Agronomy, Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia

    Widhi Dyah Sawitri

  3. Postgraduate Program in Biotechnology, University of Jember, Jember, 68121, Indonesia

    Nuriyah Inda Kurniah

  4. Department of Agricultural Microbiology, Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia

    Muhammad Saifur Rohman

  5. Faculty of Medicine, Universitas Pembangunan Nasional “Veteran” Jakarta, Jakarta, 12450, Indonesia

    Yudhi Nugraha

  6. Institute for Protein Research, Osaka University, Suita, Osaka, 565-0871, Japan

    Toshiharu Hase

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  1. Nuriyah Inda Kurniah
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Correspondence to Bambang Sugiharto.

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Kurniah, N.I., Sawitri, W.D., Rohman, M.S. et al. Mutation of UDP-glucose binding motif residues lead to increased affinity for ADP-glucose in sugarcane sucrose phosphate synthase. Mol Biol Rep 48, 1697–1706 (2021). https://doi.org/10.1007/s11033-021-06181-8

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