植物蔗糖合酶的结构、功能及应用

蔗糖合酶（Sucrose synthase, EC 2.4.1.13, SuS）是植物中广泛存在的一种糖基转移酶,能催化蔗糖的分解及合成反应,是叶片光合作用产物蔗糖进入各种代谢途径所必需的关键酶之一,在植物的生长发育过程中发挥着至关重要的作用．近年研究表明,蔗糖合酶不仅在植物淀粉合成、提高植株抗逆性和影响植株生长等方面扮演着重要的角色,也能为机体提供核苷单糖供体,而这个特性也使得蔗糖合酶基因可以作为一个催化成分被用于核苷单糖的生物合成,具有广泛的应用前景．本文对蔗糖合酶家族基因的染色体定位及功能、蔗糖合酶的结构及亚细胞定位,以及其所具有的生物学功能进行了综述,旨在为蔗糖合酶的进一步研究奠定理论基础．     

The unique nucleotide specificity of the sucrose synthase from Thermosynechococcus elongatus

Sucrose synthase catalyzes the reversible conversion of sucrose and UDP into fructose and UDP-glucose. In filamentous cyanobacteria, the sucrose cleavage direction plays a key physiological function in carbon metabolism, nitrogen fixation, and stress tolerance. In unicellular strains, the function of sucrose synthase has not been elucidated. We report a detailed biochemical characterization of sucrose synthase from Thermosynechococcus elongatus after the gene was artificially synthesized for optimal expression in Escherichia coli. The homogeneous recombinant sucrose synthase was highly specific for ADP as substrate, constituting the first one with this unique characteristic, and strongly suggesting an interaction between sucrose and glycogen metabolism.     

Diurnal fluctuations in cotton leaf carbon export, carbohydrate content, and sucrose synthesizing enzymes

In fully expanded leaves of greenhouse-grown cotton (Gossypium hirsutum L., cv Coker 100) plants, carbon export, starch accumulation rate, and carbon exchange rate exhibited different behavior during the light period. Starch accumulation rates were relatively constant during the light period, whereas carbon export rate was greater in the afternoon than in the morning even though the carbon exchange rate peaked about noon. Sucrose levels increased throughout the light period and dropped sharply with the onset of darkness; hexose levels were relatively constant except for a slight peak in the early morning. Sucrose synthase, usually thought to be a degradative enzyme, was found in unusually high activities in cotton leaf. Both sucrose synthase and sucrose phosphate synthetase activities were found to fluctuate diurnally in cotton leaves but with different rhythms. Diurnal fluctuations in the rate of sucrose export were generally aligned with sucrose phosphate synthase activity during the light period but not with sucrose synthase activity; neither enzyme activity correlated with carbon export during the dark. Cotton leaf sucrose phosphate synthase activity was sufficient to account for the observed carbon export rates; there is no need to invoke sucrose synthase as a synthetic enzyme in mature cotton leaves. During the dark a significant correlation was found between starch degradation rate and leaf carbon export. These results indicate that carbon partitioning in cotton leaf is somewhat independent of the carbon exchange rate and that leaf carbon export rate may be linked to sucrose formation and content during the light period and to starch breakdown in the dark.     

Transport and Metabolism of Sucrose versus Hexoses in Relation to Growth in Etiolated Pea Stem

Sucrose, supplied to detached pea (Pisum sativum L. var Alaska) epicotyls through cut bases, supported better growth of apical tissue than supplied glucose and/or fructose. The hexoses were converted mainly to sucrose in basal regions of the epicotyl but some moved as such through the epicotyl and accumulated at the apex (plumule) at a rate faster than sucrose. A greater proportion of the carbon derived from supplied hexoses than from sucrose was used for synthesis of ethanol-insoluble products throughout the epicotyl. By use of asymmetrically labeled sucrose, it was shown that neither hexose moiety was used preferentially for the synthesis of metabolites. Supplied sucrose moved as such only up to the region of cell elongation where it was hydrolyzed and completely equilibrated before moving into more apical regions. The results indicate that better growth with supplied sucrose than hexose could not have resulted from differential effects on cell division, more rapid uptake or transport of sucrose, enhanced wall synthesis, or cleavage by sucrose synthase. It is concluded that transported sucrose versus hexoses must undergo or evoke different reactions which affect growth in the region of cell elongation.     

Investigation of sucrose synthase from rice for the synthesis of various nucleotide sugars and saccharides

The unique character of the plant glucosyltransferase sucrosesynthase, to catalyse in vitro the synthesis and cleavage ofsucrose under appropriate conditions, can be exploited for theenzymatic synthesis of carbohydrates. The present paper describesthe potential utilization of sucrose synthase from rice forthe enzymatic synthesis of activated sugars and saccharides.In the cleavage reaction of sucrose, the nucleoside diphosphatescan be used in the order UDP > TDP > ADP > CDP >GDP to obtain the corresponding activated glucoses. In batchreactions, >90% conversion of UDP and TDP could be achieved.Substituting different di- and trisaccharides for sucrose inthe cleavage reaction with UDP 2-deoxysucrose was the most promisingsubstrate. Sucrose synthase was combined with UDP-galactose4'-epimerase and ß1–4 galactosyltransferaseto synthesize N-acetyllactosamine with in situ regenerationof UDP-glucose. In the synthesis reaction of sucrose synthase,different donor (UDP-sugars) and acceptor substrates were investigated.UDP-N-acetylglucosamine and UDP-xylose could be used in combinationwith fructose as acceptor. D-Xylulose, D-tagatose, D-lyxose,D-psicose, L-sorbose, D-mannose, L-arabinose, 1, 6 anhydroglucose,lactulose, raffinose and isomaltulose can serve as acceptorsfor UDP-glucose. N-acetyllactosamine nucleotide sugars saccharides sucrose synthase     

Nitrate activation of cytosolic protein kinases diverts photosynthetic carbon from sucrose to amino Acid biosynthesis: basis for a new concept

The regulation of carbon partitioning between carbohydrates (principally sucrose) and amino acids has been only poorly characterized in higher plants. The hypothesis that the pathway of sucrose and amino acid biosynthesis compete for carbon skeletons and energy is widely accepted. In this review, we suggest a mechanism involving the regulation of cytosolic protein kinases whereby the flow of carbon is regulated at the level of partitioning between the pathways of carbohydrate and nitrogen metabolism via the covalent modulation of component enzymes. The addition of nitrate to wheat seedlings (Triticum aestivum) grown in the absence of exogenous nitrogen has a dramatic, if transient, impact on sucrose formation and on the activities of sucrose phosphate synthase (which is inactivated) and phosphoenolpyruvate carboxylase (which is activated). The activities of these two enzymes are modulated by protein phosphorylation in response to the addition of nitrate, but they respond in an inverse fashion. Sucrose phosphate synthase in inactivated and phosphoenolpyruvate carboxylase is activated. Nitrate functions as a signal metabolite activating the cytosolic protein kinase, thereby modulating the activities of at least two of the key enzymes in assimilate partitioning and redirecting the flow of carbon away from sucrose biosynthesis toward amino acid synthesis.     

Ontogenic changes in enzymes of carbon metabolism in relation to carbohydrate status in developing mungbean reproductive structures

The content of free sugars and the activities of enzymes involved in carbon metabolism-sucrose synthase, acid and alkaline invertase, phosphoenol pyruvate carboxylase, malic enzyme and isocitrate dehydrogenase were determined during seed development in mungbean pods. A decrease in carbohydrate content of pod wall from 10 to 25 days after flowering (DAF) and a concomitant increase in the seed till 20 DAF was observed. Sucrose remained the dominant soluble sugar in the pod wall and seed. In the branch of inflorescence and pod wall, the activities of sucrose metabolizing enzymes, viz. acid and alkaline invertase, sucrose synthase (synthesis and cleavage) and sucrose phosphate synthase were higher at 5-10 DAF, whereas in seed the maximum activities of these enzymes were observed at the time of maximum seed filling stage (10-20 DAF). High activities of sucrose synthase at the time of rapid seed filling can be correlated to its sink strength. Higher activities of phosphoenol pyruvate carboxylase in the branch of inflorescence and pod wall than in seed may indicate the involvement of the fruiting structure for recapturing respired CO2. High activities of isocitrate dehydrogenase and malic enzyme in the seed at the time of rapid seed filling could provide NADPH and carbon skeletons required for the synthesis of various seed reserves.     

Measurement of enzymes related to sucrose metabolism in permeabilized Chlorella vulgaris cells

Sucrose phosphate synthase (UDP-glucose: D-fructose-6-phosphate-2-glucosyl transferase, EC 2.4.1.14), sucrose synthase (UDP-glucose: D-fructose-2-glucosyl transferase, EC 2.4.1.13) and invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) were measured in toluene permeabilized cells of Chlorella vulgaris Beijerinck. All three activities were detected at all stages of the growth curve; sucrose synthase and sucrose phosphate synthase showed a zone of maximum activity, while invertase increased with time of growth. Sucrose phosphate synthase and sucrose synthase (sucrose synthesis direction) were stimulated by divalent cations and inhibited by UDP. This inhibition could be reversed by Mg²⁺ or Mn²⁺. Sucrose phosphate synthase activity was inhibited by inorganic phosphate and was enhanced by glucose-6-phosphate, but was insensitive to sucrose. Arbutine decreased sucrose synthase activity in both directions. Sucrose cleavage was inhibited by divalent cations and by pyrophosphate. The effects on the enzyme activities of the presence of 2,4-dichlorophenoxyacetic acid (2,4-D), gibberellic acid, abscisic acid and kinetin in the growth medium were investigated. Sucrose synthase activity was practically unaffected by all plant hormones tested, except for the presence of kinetin which stimulated the activity. Sucrose phosphate synthase activity was increased by both kinetin and abscisic acid. The effect of the latter was partially reversed by the presence of gibberellic acid. 2,4-D and kinetin were potent stimulators of invertase activity.     

Contributions of sucrose synthase and invertase to the metabolism of sucrose in developing leaves : estimation by alternate substrate utilization

The relative contributions of invertase and sucrose synthase to initial cleavage of phloem-imported sucrose was calculated for sink leaves of soybean (Glycine max L. Merr cv Wye) and sugar beet (Beta vulgaris L. monohybrid). Invertase from yeast hydrolyzed sucrose 4200 times faster than 1′-deoxy-1′-fluorosucrose (FS) while sucrose cleavage by sucrose synthase from developing soybean leaves proceeded only 3.6 times faster than cleavage of FS. [¹⁴C]Sucrose and [¹⁴C]FS, used as tracers of sucrose, were transported at identical rates to developing leaves through the phloem. The rate of label incorporation into insoluble products varied with leaf age from 3.4 to 8.0 times faster when [¹⁴C]sucrose was supplied than when [¹⁴C]FS was supplied. The discrimination in metabolism was related to enzymatic discriminations against FS to calculate the relative contributions of invertase and sucrose synthase to sucrose cleavage. In the youngest soybean leaves measured, 4% of final laminar length (FLL), all cleavage was by sucrose synthase. Invertase contribution to sucrose metabolism was 47% by 7.6% FLL, increased to 54% by 11% FLL, then declined to 42% for the remainder of the import phase. In sugar beet sink leaves at 30% FLL invertase contribution to sucrose metabolism was 58%.     

Nodule-enhanced expression of a sucrose phosphate synthase gene member (MsSPSA) has a role in carbon and nitrogen metabolism in the nodules of alfalfa (Medicago sativa L.)

Sucrose phosphate synthase (SPS) catalyzes the first step in the synthesis of sucrose in photosynthetic tissues. We characterized the expression of three different isoforms of SPS belonging to two different SPS gene families in alfalfa (Medicago sativa L.), a previously identified SPS (MsSPSA) and two novel isoforms belonging to class B (MsSPSB and MsSPSB3). While MsSPSA showed nodule-enhanced expression, both MsSPSB genes exhibited leaf-enhanced expression. Alfalfa leaf and nodule SPS enzymes showed differences in chromatographic and electrophoretic migration and differences in V _max and allosteric regulation. The root nodules in legume plants are a strong sink for photosynthates with its need for ATP, reducing power and carbon skeletons for dinitrogen fixation and ammonia assimilation. The expression of genes encoding SPS and other key enzymes in sucrose metabolism, sucrose phosphate phosphatase and sucrose synthase, was analyzed in the leaves and nodules of plants inoculated with Sinorhizobium meliloti. Based on the expression pattern of these genes, the properties of the SPS isoforms and the concentration of starch and soluble sugars in nodules induced by a wild type and a nitrogen fixation deficient strain, we propose that SPS has an important role in the control of carbon flux into different metabolic pathways in the symbiotic nodules.     

Sucrose accumulation in sweet sorghum stem internodes in relation to growth

Sweet sorghum (Sorghum bicolor L. Moench) stems of different cultivars (NK 405. Keller and Tracy) reveal a different pattern of sucrose accumulation with respect to in-ternodal sugar content and distribution. The onset of sucrose storage is not necessarily associated with the reproductive stage of the plant, as was hitherto assumed, but obviously occurs after cessation of internodai elongation as was postulated for the sugarcane stem. For at least two of the three cultivars, ripening is an internode to internode process beginning at the lowermost culm parts. Intensive growth of the internodes, combined with a high hexose content in stern parenchyma, shows a strong positive correlation (r |Mg 0.94) to the activity of sucrose synthase (SuSy; EC 2.4.13), but not to invertase (EC 3.2.1.26) which is not present as soluble (neutral and acid) or cell wall-bound, salt-extractable enzyme in the three culsivars investigated. Sucrose synthase measured in sucrose cleavage and synthesis direction reveals divergent activity rates and sensitivity towards exogenously applied Mg²⁺ ions and pH. SuSy activity is connected to the increase of internodai sucrose content in so far as (1) its decline is a prerequisite for the onset of sucrose accumulation and (2) it remains at a constant low level during sucrose storage. Sucrose phosphate synthase (SPS; EC 2.4.1.14) activity in the sorghum stem is low compared to SuSy and uniformly distributed over all inter-nodes. Only source leaves of sorghum show a considerable SPS activity, but neither stem nor leaf SPS reveal a positive correlation to the increase of internodai sucrose content. Sucrose phosphate phosphatase (SPP; EC 3.1.3.24) amounts lo only 24–30% of the respective SPS activity but follows the same distribution pattern. None of the enzymes under study proves to be responsible for the extent of sucrose storage in the stem, so other phenomena such as transport processes within the stern tissue require further investigation.     

Sucrose Synthase in Wild Tomato, Lycopersicon chmielewskii, and Tomato Fruit Sink Strength

Here it is reported that sucrose synthase can be readily measured in growing wild tomato fruits (Lycopersicon chmielewskii) when suitable methods are adopted during fruit extraction. The enzyme also was present in fruit pericarp tissues, in seeds, and in flowers. To check for novel characteristics, the wild tomato fruit sucrose synthase was purified, by (NH₄)₂SO₄ fraction and chromatography with DE-32, Sephadex G-200, and PBA-60, to one major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The following characteristics were obtained: native protein relative molecular weight 380,000; subunit relative molecular weight 89,000; K_m values with: sucrose 53 millimolar, UDP 18.9 micromolar, UDP-glucose 88 micromolar, fructose 8.4 millimolar; pH optima between 6.2 to 7.3 for sucrose breakdown and 7 to 9 for synthesis; and temperature optima near 50°C. The enzyme exhibited a high affinity and a preference for uridylates. The enzyme showed more sensitivity to divalent cations in the synthesis of sucrose than in its breakdown. Sink strength in tomato fruits also was investigated in regard to sucrose breakdown enzyme activities versus fruit weight gain. Sucrose synthase activity was consistently related to increases in fruit weight (sink strength) in both wild and commercial tomatoes. Acid and neutral invertases were not, because the published invertase activity values were too variable for quantitative analyses regarding the roles of invertases in tomato fruit development. In rapidly growing fruits of both wild and commercially developed tomato plants, the activity of sucrose synthase per growing fruit, i.e. sucrose synthase peak activity X fruit size, was linearly related to final fruit size; and the activity exceeded fruit growth and carbon import rates by at least 10-fold. In mature, nongrowing fruits, sucrose synthase activities approached nil values. Therefore, sucrose synthase can serve as an indicator of sink strength in growing tomato fruits.     

网纹甜瓜发育果实糖分积累与蔗糖代谢参与酶的关系

随着网纹甜瓜果实的发育,果实中葡萄糖和果糖的含量增加,蔗糖的快速积累发生在果实发育的中后期,高蔗糖积累型果实中蔗糖积累速率明显快于低蔗糖积累型.蔗糖磷酸合成酶活性在果实发育的前期短暂下降, 而后稳步上升,在果实发育的中后期高蔗糖积累型果实中该酶的活性显著高于低蔗糖积累型果实;随着果实发育,蔗糖合成酶的分解活性降低而合成活性升高.酸性和中性转化酶在未成熟果实中活性较高,而在成熟果实中很低; 高蔗糖积累型果实中酸性转化酶活性显著低于同期低蔗糖积累型果实.合成蔗糖的酶活性小于分解蔗糖的酶活性时蔗糖几乎没有积累.根据这些结果推测,转化酶活性的下降、蔗糖磷酸合成酶活性的增加以及蔗糖合成酶分解活性的下降和合成活性的增加,是引起果实蔗糖积累的主要内在因子.     

The developmental and organ specific expression of sucrose cleaving enzymes in sugar beet suggests a transition between apoplasmic and symplasmic phloem unloading in the tap roots

Sucrose utilisation in sink tissues depend on its cleavage and is mediated by two different classes of enzymes, invertase and sucrose synthase, which determine the mechanism of phloem unloading. Cloning of two extracellular (BIN35 and BIN46) and one vacuolar invertase (BIN44) provided the basis for a detailed molecular analysis of the relative contribution of the sucrose cleaving enzymes to the sink metabolism of sugar beets (Beta vulgaris) during development. The determination of the steady state levels of mRNAs has been complemented by the analysis of the corresponding enzyme activities. The present study demonstrates an inverse regulation of extracellular invertase and sucrose synthase during tap root development indicating a transition between functional unloading pathways. Extracellular cleavage by invertase is the dominating mechanism to supply hexoses via an apoplasmic pathway at early stages of storage root development. Only at later stages sucrose synthase takes over the function of the key sink enzyme to contribute to the sink strength of the tap root via symplasmic phloem unloading. Whereas mRNAs for both extracellular invertase BIN35 and sucrose synthase were shown to be induced by mechanical wounding of mature leaves of adult plants, only sucrose synthase mRNA was metabolically induced by glucose in this source organ supporting the metabolic flexibility of this species.     

Regulation of photosynthetic carbon metabolism in cucumber by light intensity and photosynthetic period

The effects of photosynthetic periods and light intensity on cucumber (Cucumis sativus L.) carbon exchange rates and photoassimilate partitioning were determined in relation to the activities of galactinol synthase and sucrose-phosphate synthase. Carbon assimilation and partitioning appeared to be controlled by different mechanisms. Carbon exchange rates were influenced by total photon flux density, but were nearly constant over the entire photoperiod for given photoperiod lengths. Length of the photosynthetic periods did influence photoassimilate partitioning. Assimilate export rate was decreased by more than 60% during the latter part of the short photoperiod treatment. This decrease in export rate was associated with a sharp increase in leaf starch acccumulation rate. Results were consistent with the hypothesis that starch accumulation occurs at the expense of export under short photoperiods. Galactinol synthase activities did not appear to influence the partitioning of photoassimilates between starch and transport carbohydrates. Sucrose phosphate synthase activities correlated highly with sugar formation rates (sucrose, raffinose, stachyose + assimilate export rate, r = 0.93, α = 0.007). Cucumber leaf sucrose phosphate synthase fluctuated diurnally in a similar pattern to that observed in vegetative soybean plants.     

Regulation of Spinach Leaf Sucrose Phosphate Synthase by Glucose-6-Phosphate, Inorganic Phosphate, and pH

Sucrose phosphate synthase was partially purified from spinach leaves and the effects and interactions among glucose-6-P, inorganic phosphate (Pi), and pH were investigated. Glucose-6-P activated sucrose phosphate synthase and the concentration required for 50% of maximal activation increased as the concentration of fructose-6-P was decreased. Inorganic phosphate inhibited sucrose phosphate synthase activity and antagonized the activation by glucose-6-P. Inorganic phosphate caused a progressive increase in the concentration of glucose-6-P required for 50% maximal activation from 0.85 mm (minus Pi) to 9.9 mm (20 mm Pi). In the absence of glucose-6-P, Pi caused partial inhibition of sucrose phosphate synthase activity (about 65%). The concentration of Pi required for 50% maximal inhibition decreased with a change in pH from 6.5 to 7.5. When the effect of pH on Pi ionization was taken into account, it was found that per cent inhibition increased hyperbolically with increasing dibasic phosphate concentration independent of the pH. Sucrose phosphate synthase had a relatively broad pH optimum centered at pH 7.5. Inhibition by Pi was absent at pH 5.5, but became more pronounced at alkaline pH, whereas activation by glucose-6-P was observed over the entire pH range tested. The results suggested that glucose-6-P and Pi bind to sites distinct from the catalytic site, e.g. allosteric sites, and that the interactions of these effectors with pH and concentrations of substrate may be involved in the regulation of sucrose synthesis in vivo.     

Carbon partitioning to cellulose synthesis

This article discusses the importance and implications of regulating carbon partitioning to cellulose synthesis, the characteristics of cells that serve as major sinks for cellulose deposition, and enzymes that participate in the conversion of supplied carbon to cellulose. Cotton fibers, which deposit almost pure cellulose into their secondary cell walls, are referred to as a primary model system. For sucrose synthase, we discuss its proposed role in channeling UDP-Glc to cellulose synthase during secondary wall deposition, its gene family, its manipulation in transgenic plants, and mechanisms that may regulate its association with sites of polysaccharide synthesis. For cellulose synthase, we discuss the organization of the gene family and how protein diversity could relate to control of carbon partitioning to cellulose synthesis. Other enzymes emphasized include UDP-Glc pyrophosphorylase and sucrose phosphate synthase. New data are included on phosphorylation of cotton fiber sucrose synthase, possible regulation by Ca²⁺ of sucrose synthase localization, electron microscopic immunolocalization of sucrose synthase in cotton fibers, and phylogenetic relationships between cellulose synthase proteins, including three new ones identified in differentiating tracheary elements of Zinnia elegans. We develop a model for metabolism related to cellulose synthesis that implicates the changing intracellular localization of sucrose synthase as a molecular switch between survival metabolism and growth and/or differentiation processes involving cellulose synthesis. Abbreviations: CesA, cellulose synthase; Csl, cellulose-like synthase (genes); DCB, dichlobenil; DPA, days after anthesis; SPS, sucrose phosphate synthase; SuSy, sucrose synthase; P-SuSy, particulate SuSy; S-SuSy, soluble SuSy     

Sucrose synthase of soybean nodules

Sucrose synthase (UDPglucose: d-fructose 2-α-d-glucosyl transferase, EC 2.4.1.13) has been purified from the plant cytosolic fraction of soybean (Glycine max L. Merr cv Williams) nodules. The native enzyme had a molecular weight of 400,000. The subunit molecular weight was 90,000 and a tetrameric structure is proposed for soybean nodule sucrose synthase. Optimum activity in the sucrose cleavage and synthesis directions was at pH 6 and pH 9.5 respectively, and the enzyme displayed typical Michaelis-Menten kinetics. Soybean nodule sucrose synthase had a high affinity for UDP (K_m, 5 micromolar) and a relatively low affinity for ADP (apparent K_m, 0.13 millimolar) and CDP (apparent K_m, 1.1 millimolar). The K_m for sucrose was 31 millimolar. In the synthesis direction, UDPglucose (K_m, 0.012 millimolar) was a more effective glucosyl donor than ADPglucose (K_m, 1.6 millimolar) and the K_m for fructose was 3.7 millimolar. Divalent cations stimulated activity in both the cleavage and synthesis directions and the enzyme was very sensitive to inhibition by heavy metals.