title: Purification and characterization of recombinant sugarcane sucrose phosphate synthase expressed in E. coli and insect Sf9 cells: an importance of the N-terminal domain for an allosteric regulatory property.
abstract: Sucrose phosphate synthase (SPS) catalyses the transfer of glycosyl group of uridine diphosphate glucose to fructose-6-phosphate to form sucrose-6-phosphate. Plant SPS plays a key role in photosynthetic carbon metabolisms, which activity is modulated by an allosteric activator glucose-6-phosphate (G6P). We produced recombinant sugarcane SPS using Escherichia coli and Sf9 insect cells to investigate its structure-function relationship. When expressed in E. coli, two forms of SPS with different sizes appeared; the larger was comparable in size with the authentic plant enzyme and the shorter was trimmed the N-terminal 20 kDa region off. In the insect cells, only enzyme with the authentic size was produced. We purified the trimmed SPS and the full size enzyme from insect cells and found their enzymatic properties differed significantly; the full size enzyme was activated allosterically by G6P, while the trimmed one showed a high activity even without G6P. We further introduced a series of N-terminal truncations up to 171 residue and found G6P-independent activity was enhanced by the truncation. These combined results indicated that the N-terminal region of sugarcane SPS is crucial for the allosteric regulation by G6P and may function like a suppressor domain for the enzyme activity.
authors.affiliation: Research Center for State-of-the-Art Functional Protein Analysis, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan;
authors.forename: Widhi Dyah
authors.initials: WD
authors.lastname: Sawitri
authors.affiliation: Research Center for State-of-the-Art Functional Protein Analysis, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan;
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Narita
authors.affiliation: Research Center for State-of-the-Art Functional Protein Analysis, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan;
authors.forename: Etsuko
authors.initials: E
authors.lastname: Ishizaka-Ikeda
authors.affiliation: Center for Development of Advanced Science and Technology, Jember University, Jl. Kalimantan No. 37, Kampus Tegalboto, Jember 68121, Indonesia; and sugiharto.fmipa@unej.ac.id.
authors.forename: Bambang
authors.initials: B
authors.lastname: Sugiharto
authors.affiliation: Division of Protein Chemistry, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan enzyme@protein.osaka-u.ac.jp.
authors.forename: Toshiharu
authors.initials: T
authors.lastname: Hase
authors.affiliation: Research Center for State-of-the-Art Functional Protein Analysis, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan;
authors.forename: Atsushi
authors.initials: A
authors.lastname: Nakagawa
mesh_terms: D000818:Animals; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005964:Glucosyltransferases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D031786:Saccharum / Q000201:enzymology* / Q000235:genetics; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C022698:sucrose-phosphate synthase
keywords: allosteric regulation; carbon metabolism; recombinant enzyme; sucrose phosphate synthase; sugarcane
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