酵母菌 Pichia pastoris 中表現重組水稻蔗糖合成酶 RSuS3 之性質與結構探討

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酵母菌 Pichia pastoris 中表現重組水稻蔗糖合成酶 RSuS3 之性質與結構探討

Studies on the Biochemical and Structural Characteristics of Recombinant Rice Sucrose Synthase RSuS3 in Pichia pastoris

蔡逸君 , Masters　　Advisor：王愛玉　　Co-advisor ：宋賢一;楊啟伸

繁體中文

水稻蔗糖合成酶； Sucrose synthase ； SuS ； Pichia pastoris

Abstract │ Reference (140) │ Times Cited (4) │ Altmetrics

Abstract 〈TOP〉

Parallel Abstract 〈TOP〉

Sucrose synthase catalyzes the reversible conversion of sucrose and UDP into fructose and UDPG.  Among the three isogenes in rice (RSus1, RSus2, RSus3), the gene products of RSus1 and RSus2 are ubiquitously present in all rice tissues, while the expression of RSus3 is seed-specific.  In this research, recombinant RSuS3 expressed from Pichia pastoris is used to study on the biological and structural characteristics of RSuS3.
     Expression plasmid containing RSus3 cDNA transformed into yeast P. pastoris for expression, and the recombinant product is purified to analyze its properties.  The results show that the major activity of recombinant RSuS3 is to generate sucrose from fructose and UDPG, while the sucrose-cleavage activity of RSuS3 is very low.  Besides, Mg2+ is able to promote the sucrose-synthesis activity of RSuS3.  The presence of sugar metabolites have no significant effect on recombinant RSuS3 activity, neither do the presence of phospholipids. 
     There are eight cysteines on the surface of recombinant RSuS3, and the native protein is present as a tetramer as revealed from the result of transmission electron microscopy (TEM).  Furthermore, native crystal, heavy atom co-crystal and diffraction data of native crystal at 3.53 &Aring; resolution has been obtained as the initial results of first step of X-ray crystallography.

Reference ( 140 ) 〈TOP〉

Amor, Y., Haigler, C.H., Johnson, S., Wainescott, M. and Delmer, D.P. (1995) A membrane-associated form of sucrose synthase and its potential role in synthesis of cellulose and callose in plants. Proceedings of the National Academy of Sciences of the United States of America 92, 9353-9357.
連結：
Anguenot, R., Yelle, S. and Nguyen-Quoc, B. (1999) Purification of tomato sucrose synthase phosphorylated isoforms by Fe(III)-immobilized metal affinity chromatography. Archives of Biochemistry and Biophysics 365, 163-169.
連結：
Baroja-Fernandez, E., Munoz, F.J., Saikusa, T., Rodriguez-Lopez, M., Akazawa, T. and Pozueta-Romero, J. (2003) Sucrose synthase catalyzes the de novo production of ADPglucose linked to starch biosynthesis in heterotrophic tissues of plants. Plant and Cell Physiology 44, 500-509.
連結：
Barratt, D.H., Barber, L., Kruger, N.J., Smith, A.M., Wang, T.L. and Martin, C. (2001) Multiple, distinct isoforms of sucrose synthase in pea. Plant Physiology 127, 655-664.
連結：
Baud, S., Vaultier, M.N. and Rochat, C. (2004) Structure and expression profile of the sucrose synthase multigene family in Arabidopsis. Journal of Experimental Botany 55, 397-409.
連結：

Times Cited (4) 〈TOP〉

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