Purification and characterization of recombinant sugarcane sucrose phosphate synthase expressed inE. coliand 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 20kDa 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.Keywords: allosteric regulation/carbon metabolism/ recombinant enzyme/sucrose phosphate synthase/ sugarcane.Abbreviations: F6P, fructose-6-phophate; G6P, glucose-6-phosphate SPS, sucrose phosphate synthase; UDP-G, uridine diphosphate glucose.Sucrose is the primary photosynthate in plant leaves and transported to various growing tissues and storage organs for allocation of carbon resources through whole plant body. Sucrose phosphate synthase (SPS) plays a key role in the pathway of sucrose synthesis, which catalyses formation of sucrose-6-phosphate (S6P) by use of fructose-6-phosphate (F6P) and uridine diphosphate-glucose (UDP-G) as substrates (13). The activity of SPS is regulated by diurnal cycles of day/ night (4) and other environmental factors such as osmotic stress (5) and cold temperature (6). Protein phosphorylation of certain serine residues of SPS polypeptide and allosteric control by metabolites, such as glucose-6-phospate (G6P) and inorganic phosphate, are involved in the regulation of SPS activity (7,8). Such molecular characteristics of SPS are believed to be crucial for playing the physiological role of regulating photosynthetic carbon flux into sucrose.Plant SPSs have a molecular mass around 120 kDa and consists of three domains. The central region contains glycosyltransferase domain responsible for the catalytic function of SPS (9). The C-terminal region resembles sucrose phosphate phosphatase and the N-terminal region has no clear similarity with any other proteins. The SPSs from the photosynthetic cyanobacteria (10) and non-photosynthetic bacteria (11) lack the N-terminal domain or both terminal domains of plant SPS, respectively, and unlike plant SPS, their enzyme activity is not allosterically regulated (12,13). This in...

Overexpression of Sucrose Phosphate Synthase Enhanced Sucrose Content and Biomass Production in Transgenic Sugarcane

“…Thus, it is postulated that the manipulation of sucrose transporter genes, as well as SWEET expression, in cooperation with increased SPS activity, might further increase sucrose concentration in the stalks of sugarcane. In addition, it was recently reported that N-terminal truncated SPS shows higher activity, avoiding regulation by allosteric effectors [42]. Future research will aim at further increasing sucrose accumulation in plants using the N-terminal deleted SPS.…”

“…SPS activity was assayed by measuring the formation of sucrose-6-phosphate in the desalted extract as previously described [42]. The assay mixture (70 µL) contained 30 mM MOPS-NaOH (pH 7.5), 10 mM MgCl 2 , 15 mM UDP-glucose, 10 mM fructose-6-phosphate (F6P), and 10 mM glucose-6-phosphate (G6P).…”

“…The analysis was conducted by separating the proteins from the crude extract using SDS-PAGE (12.5% polyacrylamide) and transferring them onto the Immobilon-P transfer membrane (Thermo Scientific, Rockford, IL, USA) using a semi-dry trans-blotter (Bio-Rad, Irvine, CA, USA). The membrane was then separately incubated with polyclonal antibodies against PEPC, Rubisco [46], or recombinant SPS1 proteins [42], and then diluted in tris-buffered saline (TBS) containing 0.5% skim milk overnight. After washing with TBS, the membrane was incubated with a secondary antibody of goat anti-rabbit IgG Alkaline phosphatase conjugate (Bio-Rad, Irvine, CA, USA) at 1:3000 dilution for 60 min.…”

TRANSFORMASI GENETIK DAN EKSPRESI MUTAN <em>SUCROSE PHOSPHATE SYNTHASE</em> PADA TANAMAN TOMAT

“…Untuk mempelajari peran domain N-terminus pada regulasi alosterik SPS, maka mutan SPS dengan pemotongan domain N-terminus dianalisis secara in vitro. Hasil analisis tersebut didapatkan bahwa SPS yang kehilangan domain N-terminus tidak dapat diaktivasi oleh G6P, tetapi spesifik aktifitas enzim meningkat hingga 10 kali lipat (Sawitri et al 2016). Meskipun mutan tersebut lebih stabil dan memiliki prospek dalam pengembangan bioteknologi, tetapi karakterisasi dan ekspresi penghilangan domain N-terminus SPS perlu dikaji lebih lanjut pada tanaman.…”

Detection and Response of Sugarcane against the Infection of Sugarcane Mosaic Virus (SCMV) in Indonesia

“…Quantitatively, SPS activity was estimated by following Sawitri et al [23]. Leaf extract was cleaned up using Sephadex G-25 and subjected to an enzyme activity assay.…”