TY - JOUR A1 - Ruzanski, Christian A1 - Smirnova, Julia A1 - Rejzek, Martin A1 - Cockburn, Darrell A1 - Pedersen, Henriette L. A1 - Pike, Marilyn A1 - Willats, William G. T. A1 - Svensson, Birte A1 - Steup, Martin A1 - Ebenhöh, Oliver A1 - Smith, Alison M. A1 - Field, Robert A. T1 - A bacterial glucanotransferase can replace the complex maltose metabolism required for starch to sucrose conversion in leaves at night JF - The journal of biological chemistry N2 - Controlled conversion of leaf starch to sucrose at night is essential for the normal growth of Arabidopsis. The conversion involves the cytosolic metabolism of maltose to hexose phosphates via an unusual, multidomain protein with 4-glucanotransferase activity, DPE2, believed to transfer glucosyl moieties to a complex heteroglycan prior to their conversion to hexose phosphate via a cytosolic phosphorylase. The significance of this complex pathway is unclear; conversion of maltose to hexose phosphate in bacteria proceeds via a more typical 4-glucanotransferase that does not require a heteroglycan acceptor. It has recently been suggested that DPE2 generates a heterogeneous series of terminal glucan chains on the heteroglycan that acts as a glucosyl buffer to ensure a constant rate of sucrose synthesis in the leaf at night. Alternatively, DPE2 and/or the heteroglycan may have specific properties important for their function in the plant. To distinguish between these ideas, we compared the properties of DPE2 with those of the Escherichia coli glucanotransferase MalQ. We found that MalQ cannot use the plant heteroglycan as an acceptor for glucosyl transfer. However, experimental and modeling approaches suggested that it can potentially generate a glucosyl buffer between maltose and hexose phosphate because, unlike DPE2, it can generate polydisperse malto-oligosaccharides from maltose. Consistent with this suggestion, MalQ is capable of restoring an essentially wild-type phenotype when expressed in mutant Arabidopsis plants lacking DPE2. In light of these findings, we discuss the possible evolutionary origins of the complex DPE2-heteroglycan pathway. KW - Carbohydrate Metabolism KW - Computer Modeling KW - Metabolic Regulation KW - Oligosaccharide KW - Plant Biochemistry KW - Glucanotransferase KW - Leaf Cell KW - Maltose Metabolism KW - Starch Degradation Y1 - 2013 U6 - https://doi.org/10.1074/jbc.M113.497867 SN - 0021-9258 SN - 1083-351X VL - 288 IS - 40 SP - 28581 EP - 28598 PB - American Society for Biochemistry and Molecular Biology CY - Bethesda ER - TY - JOUR A1 - Comparot-Moss, Sylviane A1 - Koetting, Oliver A1 - Stettler, Michaela A1 - Edner, Christoph A1 - Graf, Alexander A1 - Weise, Sean E. A1 - Streb, Sebastian A1 - Lue, Wei-Ling A1 - MacLean, Daniel A1 - Mahlow, Sebastian A1 - Ritte, Gerhard A1 - Steup, Martin A1 - Chen, Jychian A1 - Zeeman, Samuel C. A1 - Smith, Alison M. T1 - A putative phosphatase, LSF1, is required for normal starch turnover in Arabidopsis leaves N2 - A putative phosphatase, LSF1 (for LIKE SEX4; previously PTPKIS2), is closely related in sequence and structure to STARCH-EXCESS4 (SEX4), an enzyme necessary for the removal of phosphate groups from starch polymers during starch degradation in Arabidopsis (Arabidopsis thaliana) leaves at night. We show that LSF1 is also required for starch degradation: lsf1 mutants, like sex4 mutants, have substantially more starch in their leaves than wild-type plants throughout the diurnal cycle. LSF1 is chloroplastic and is located on the surface of starch granules. lsf1 and sex4 mutants show similar, extensive changes relative to wild-type plants in the expression of sugar-sensitive genes. However, although LSF1 and SEX4 are probably both involved in the early stages of starch degradation, we show that LSF1 neither catalyzes the same reaction as SEX4 nor mediates a sequential step in the pathway. Evidence includes the contents and metabolism of phosphorylated glucans in the single mutants. The sex4 mutant accumulates soluble phospho- oligosaccharides undetectable in wild-type plants and is deficient in a starch granule-dephosphorylating activity present in wild-type plants. The lsf1 mutant displays neither of these phenotypes. The phenotype of the lsf1/sex4 double mutant also differs from that of both single mutants in several respects. We discuss the possible role of the LSF1 protein in starch degradation. Y1 - 2010 UR - http://www.plantphysiol.org/ U6 - https://doi.org/10.1104/pp.109.148981 SN - 0032-0889 ER - TY - JOUR A1 - Koetting, Oliver A1 - Santelia, Diana A1 - Edner, Christoph A1 - Eicke, Simona A1 - Marthaler, Tina A1 - Gentry, Matthew S. A1 - Comparot-Moss, Sylviane A1 - Chen, Jychian A1 - Smith, Alison M. A1 - Steup, Martin A1 - Ritte, Gerhard A1 - Zeeman, Samuel C. T1 - STARCH-EXCESS4 is a laforin-like phosphoglucan phosphatase required for starch degradation in Arabidopsis thaliana N2 - Starch is the major storage carbohydrate in plants. It is comprised of glucans that form semicrystalline granules. Glucan phosphorylation is a prerequisite for normal starch breakdown, but phosphoglucan metabolism is not understood. A putative protein phosphatase encoded at the Starch Excess 4 (SEX4) locus of Arabidopsis thaliana was recently shown to be required for normal starch breakdown. Here, we show that SEX4 is a phosphoglucan phosphatase in vivo and define its role within the starch degradation pathway. SEX4 dephosphorylates both the starch granule surface and soluble phosphoglucans in vitro, and sex4 null mutants accumulate phosphorylated intermediates of starch breakdown. These compounds are linear alpha-1,4-glucans esterified with one or two phosphate groups. They are released from starch granules by the glucan hydrolases alpha-amylase and isoamylase. In vitro experiments show that the rate of starch granule degradation is increased upon simultaneous phosphorylation and dephosphorylation of starch. We propose that glucan phosphorylating enzymes and phosphoglucan phosphatases work in synergy with glucan hydrolases to mediate efficient starch catabolism. Y1 - 2009 UR - http://www.plantcell.org/ U6 - https://doi.org/10.1105/tpc.108.064360 SN - 1040-4651 ER - TY - JOUR A1 - Yu, Tien-Shin A1 - Kofler, Heike A1 - Häusler, Rainer E. A1 - Hille, Diana A1 - Flügge, Ulf-Ingo A1 - Zeeman, Samuel C. A1 - Smith, Alison M. A1 - Kossmann, Jens A1 - Lloyd, James R. A1 - Ritte, Gerhard A1 - Steup, Martin A1 - Lue, Wei-Ling A1 - Chen, Jychian A1 - Weber, Andreas P. M. T1 - The Arabidopsis sex1 mutant is defective in the R1 protein, a general regulator of starch degradation in plants, and not in the chloroplast hexose transporter Y1 - 2001 SN - 1040-4651 ER - TY - JOUR A1 - Fettke, Jörg A1 - Chia, Tansy A1 - Eckermann, Nora A1 - Smith, Alison M. A1 - Steup, Martin T1 - A transglucosidase necessary for starch degradation and maltose metabolism in leaves at night acts on cytosolic heteroglycans (SHG) N2 - The recently characterized cytosolic transglucosidase DPE2 (EC 2.4.1.25) is essential for the cytosolic metabolism of maltose, an intermediate on the pathway by which starch is converted to sucrose at night. In in vitro assays, the enzyme utilizes glycogen as a glucosyl acceptor but the in vivo acceptor molecules remained unknown. In this communication we present evidence that DPE2 acts on the recently identified cytosolic water-soluble heteroglycans (SHG) as does the cytosolic phosphorylase (EC 2.4.1.1) isoform. By using in vitro two-step C-14 labeling assays we demonstrate that the two transferases can utilize the same acceptor sites of the SHG. Cytosolic heteroglycans from a DPE2-deficient Arabidopsis mutant were characterized. Compared with the wild type the glucose content of the heteroglycans was increased. Most of the additional glucosyl residues were found in the outer chains of SHG that are released by an endo- alpha-arabinanase (EC 3.2.1.99). Additional starch-related mutants were characterized for further analysis of the increased glucosyl content. Based on these data, the cytosolic metabolism of starch-derived carbohydrates is discussed Y1 - 2006 UR - http://www3.interscience.wiley.com/cgi-bin/issn?DESCRIPTOR=PRINTISSN&VALUE=0960-7412 U6 - https://doi.org/10.1111/j.1365-313X.2006.02732.x SN - 0960-7412 ER -