Karakterisasi Fungsi dan Peran Domain Enzim Sucrose Phosphate Synthase Tebu Melalui Studi Delesi Mutagenesis

https://doi.org/10.22146/veg.81130

Widhi Dyah Sawitri(1*), Bambang Sugiharto(2)

(1) Fakultas Pertanian, Universitas Gadjah Mada
(2) Jurusan Biologi, FMIPA, Universitas Jember
(*) Corresponding Author

Abstract


Sucrose phosphate synthase (SPS) berperan penting untuk biosintesis sukrosa pada tanaman. SPS tanaman dibagi menjadi beberapa domain diantaranya bagian domian N-terminal, domain C-terminal, dan domain glycosyltransferase (GT). Namun, fungsi domain N-terminal dan C-terminal masih belum jelas. Oleh karenanya, dilakukan studi karakterisasi fungsi masing-masing domain SPS melalui penghilangan domain N-terminal (∆N), C-terminal (∆C), dan kedua domain tersebut (∆NC). Mutan ∆N, ∆C, dan ∆NC diekspresikan melalui sistem rekombinan E. coli dan dilakukan purifikasi protein secara parsial untuk menghilangkan sebagian protein kontaminan. Protein rekombinan mutan dikonfirmasi ekspresinya melalui teknik western blot dan dilakukan uji aktivitas enzim. Hasil studi menunjukkan bahwa ekspresi dan aktivitas enzim mutan ∆N meningkat. Sebaliknya, pada mutan ∆C dan ∆NC terjadi penurunan aktivitas enzim, bahkan mendekati kondisi tidak aktif. Hal ini mengindikasikan bahwa bagian domain C-terminal SPS tanaman memiliki peran yang sangat krusial untuk sintesis sukrosa. Melalui studi ini dapat diketahui bahwa domain N-terminal diindikasikan berperan dalam regulasi alosterik enzim, sedangkan domain C-terminal diasumsikan berkaitan dengan reaksi katalitik enzim.

Keywords


sucrose phosphate synthase (SPS); mutasi delesi; metabolisme sukrosa; tebu

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References

Afidah, S.N., I.D. Agustien, P. Dewanti, and B. Sugiharto, 2021. Increased activity of sugarcane sucrose-phosphate synthase in transgenic tomato in response to N-terminal truncation. Indonesian Journal of Biotechnology, 27(1), pp.43-50.

Anur, R.M., N. Mufithah, W.D. Sawitri, H. Sakakibara, and B. Sugiharto. 2020. Overexpression of sucrose phosphate synthase enhanced sucrose content and biomass production in transgenic sugarcane. Plants, 9(200), pp.1-11.

Chua, T.K., J.M. Bujnicki, T.C. Tan, F. Huynh, B.K. Patel, and Sivaraman. 2008. The structure of sucrose phosphate synthase from Halothermothrix orenii reveals its mechanism of action and binding mode. The Plant Cell, 20, pp.1059-1072.

Fibriani, S., I.D. Agustien, W.D. Sawitri, dan B. Sugiharto. 2019. Transformasi genetik dan ekspresi mutan sucrose-phosphate synthase pada tanaman tomat. Jurnal Bioteknologi dan Biosains Indonesia, 6(1), pp. 130-138.

Haigler, C.H., B. Singh, D. Zhang, S. Hwang, C. Wu, W.X. Cai, M. Hozain, W. Kang, B. Kiedaisch, R.E. Strauss, E.F. Hequet, B.G. Wyatt, G.M. Jividen, and A.S. Holaday, 2007. Transgenic cotton over-producing spinach sucrose phosphate synthase showed enchanced leaf sucrose synthesis and improved fiber quality under controlled environmental conditions. Plant Mol Biol, 63, pp. 815-832.

Huber, S.C., and J.L. Huber. 1996. Role and regulation of sucrose-phosphate synthase in higher plants. Annu Rev Plant Physiol Plant Mol Bio, 47, pp. 431-44.

Ishimaru, K., N. Hirotsu, T. Kashiwagi, Y. Madoka, K. Nagasuga, K. Ono, and R. Ohsugi. 2008. Overexpression of a maiza SPS gene improves yield characters of potato under field conditions. Plant Prod Sci, 11(1), pp. 104-107.

Kurniah, N.I., W.D. Sawitri, M.S. Rohman, Y. Nugraha, T. Hase, and B. Sugiharto. 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.

Lemoine, R., S. La Camera, R. Atanassova, F. Dedaldechamp, T. Allario, N. Pourtau, J.L. Bonnemain, M. Laloi, P. Coutos-Thevenot, L. Maurousset, M. Faucher, C. Girousse, P. Lemonnier, J. Parrilla, and M. Durand. 2013. Source-to-sink transport of sugar and regulation by environmental factors. Front Plant Sci, 272(4), pp. 1-21.

Li, Y., Y. Yao, G. Yang, J. Tang, G.J. Ayala, X. Li, W. Zhang, Q. Han, T. Yang, H. Wang, K.H. Mayo, and J. Su. 2020. Co-crystal structure of Thermosynechococcus elongatus sucrose phosphate synthase with UDP and sucrose-6-phosphate provides insight into its mechanism of action involving and oxocarbenium ion and the glycosidic bond. Front Microbiol, 1050(11), pp. 1-15.

Maloney, V.J., J.Y. Park, F. Unda, and S.D. Mansfield. 2015. Sucrose-phosphate synthase and sucrose phosphate phosphatase interact in planta and promote plant growth and biomass accumulation. Journal of Experimental Botany, 66(14), pp. 4383-4394.

Ruan, Y.L., 2012. Signaling role of sucrose metabolism in development. Molecular plant, 5(4), pp. 763-765.

Sawitri, W.D., H. Narita, E. Ishizaka-Ikeda, B. Sugiharto, T. Hase, and A. Nakagawa. 2016. Purification and characterization of recombinant sugarcane sucrose phosphate synthase expressed in E. coli and insect Sf9 cells: and importance of the N-terminal domain for an allosteric regulatory property. J. Biochem, 159(6), pp. 599-607.

Sawitri, W.D., S.N. Afidah, A. Nakagawa, T. Hase, and B. Sugiharto. 2018. Identification of UDP-glucose binding site in glycosyltransferase domain of sucrose phosphate synthase from sugarcane (Saccharum officinarum) by structure-based site-directed mutagenesis. Biophysical Reviews, 10, pp. 293-298.

Sugiharto, B., H. Sakakibara, Sumadi, and T. Sugiyama. 1997. Differential expression of two genes for sucrose-phosphate synthase sugarcane: molecular cloning of the cDNAs and comparative analysis of gene expression. Plant Cell Physiol, 38(8), pp. 961-965.



DOI: https://doi.org/10.22146/veg.81130

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