Single-celled apicomplexan parasites are known to cause major diseases in humans and animals including malaria, toxoplasmosis, and coccidiosis. The presence of apicoplasts with the remnant of a plastid-like DNA argues that these parasites evolved from photosynthetic ancestors possibly related to the dinoflagellates. Toxoplasma gondii displays amylopectin-like polymers within the cytoplasm of the dormant brain cysts. Here we report a detailed structural and comparative analysis of the Toxoplasma gondii, green alga Chlamydomonas reinhardtii, and dinoflagellate Crypthecodinium cohnii storage polysaccharides. We show Toxoplasma gondii amylopectin to be similar to the semicrystalline floridean starch accumulated by red algae. Unlike green plants or algae, the nuclear DNA sequences as well as biochemical and phylogenetic analysis argue that the Toxoplasma gondii amylopectin pathway has evolved from a totally different UDP-glucose-based metabolism similar to that of the floridean starch accumulating red alga Cyanidioschyzon merolae and, to a lesser extent, to those of glycogen storing animals or fungi. In both red algae and apicomplexan parasites, isoamylase and glucan-water dikinase sequences are proposed to explain the appearance of semicrystalline starch-like polymers. Our results have built a case for the separate evolution of semicrystalline storage polysaccharides upon acquisition of photosynthesis in eukaryotes.
SummaryAmong the three distinct starch phosphorylase activities detected in Chlamydomonas reinhardtii, two distinct plastidial enzymes (PhoA and PhoB) are documented while a single extraplastidial form (PhoC) displays a higher affinity for glycogen as in vascular plants. The two plastidial phosphorylases are shown to function as homodimers containing two 91-kDa (PhoA) subunits and two 110-kDa (PhoB) subunits. Both lack the typical 80-amino-acid insertion found in the higher plant plastidial forms. PhoB is exquisitely sensitive to inhibition by ADP-glucose and has a low affinity for malto-oligosaccharides. PhoA is more similar to the higher plant plastidial phosphorylases: it is moderately sensitive to ADP-glucose inhibition and has a high affinity for unbranched malto-oligosaccharides. Molecular analysis establishes that STA4 encodes PhoB. Chlamydomonas reinhardtii strains carrying mutations at the STA4 locus display a significant decrease in amounts of starch during storage that correlates with the accumulation of abnormally shaped granules containing a modified amylopectin structure and a high amylose content. The wild-type phenotype could be rescued by reintroduction of the cloned wild-type genomic DNA, thereby demonstrating the involvement of phosphorylase in storage starch synthesis.
Plant ␣-1,4 glucanotransferases (disproportionating enzymes, or D-enzymes) transfer glucan chains among oligosaccharides with the concomitant release of glucose (Glc). Analysis of Chlamydomonas reinhardtii sta11-1 mutants revealed a correlation between a D-enzyme deficiency and specific alterations in amylopectin structure and starch biosynthesis, thereby suggesting previously unknown biosynthetic functions. This study characterized the biochemical activities of the ␣-1,4 glucanotransferase that is deficient in sta11-1 mutants. The enzyme exhibited the glucan transfer and Glc production activities that define D-enzymes. D-enzyme also transferred glucans among the outer chains of amylopectin (using the polysaccharide chains as both donor and acceptor) and from malto-oligosaccharides into the outer chains of either amylopectin or glycogen. In contrast to transfer among oligosaccharides, which occurs readily with maltotriose, transfer into polysaccharide required longer donor molecules. All three enzymatic activities, evolution of Glc from oligosaccharides, glucan transfer from oligosaccharides into polysaccharides, and transfer among polysaccharide outer chains, were evident in a single 62-kD band. Absence of all three activities co-segregated with the sta11-1 mutation, which is known to cause abnormal accumulation of oligosaccharides at the expense of starch. To explain these data we propose that D-enzymes function directly in building the amylopectin structure.In plants, the only ␣-1,4 glucanotransferases reported to be present at the time of starch synthesis are collectively called D-enzymes (Peat et al., 1956;Takaha et al., 1993). D-enzymes act on soluble oligosaccharides at least three Glc residues long (maltotriose) and disproportionate them into oligosaccharides of various lengths at the expense of Glc formation. In such a reaction an ␣-1,4 linkage is cleaved from a donor unbranched oligosaccharide of at least three Glc residues, and the resulting chain segment is transferred to another acceptor glucan, creating a novel ␣-1,4 linkage. Maltosyl residues are often transferred as a result of D-enzyme action, but maltose itself is not a product of the reaction. However, both Glc and maltose can be used as acceptors (Jones and Whelan, 1969). It is known that in Arabidopsis leaves, D-enzyme is the major maltotriosemetabolizing enzyme present (Lin and Preiss, 1988;Zeeman et al., 1998). The presence of this activity during potato tuber development has led investigators to suggest that D-enzyme might be required for some specific aspect of starch biosynthesis (Takaha et al., 1993). Like a few other glucanotransferases, such as branching enzyme (Takaha et al., 1996a), D-enzyme has been recently shown to lead to the formation of cyclic compounds after prolonged incubation of both amylose and amylopectin with high amounts of pure activity (Takaha et al., 1996b(Takaha et al., , 1998. It is not known if this property relates to the physiological function of this enzyme. D-enzyme is believed to be part of the starch degra...
(A.M.M.) Chlamydomonas reinhardtii mutants of the STA8 gene produce reduced amounts of high amylose starch and phytoglycogen. In contrast to the previously described phytoglycogen-producing mutants of C. reinhardtii that contain no residual isoamylase activity, the sta8 mutants still contained 35% of the normal amount of enzyme activity. We have purified this residual isoamylase and compared it with the wild-type C. reinhardtii enzyme. We have found that the high-mass multimeric enzyme has reduced its average mass at least by one-half. This coincides with the disappearance of two out of the three activity bands that can be seen on zymogram gels. Wild-type and mutant enzymes are shown to be located within the plastid. In addition, they both act by cleaving off the outer branches of polysaccharides with no consistent difference in enzyme specificity. Because the mutant enzyme was demonstrated to digest phytoglycogen to completion in vitro, we propose that its inability to do so in vivo supports a function of the enzyme complex architecture in the processing of pre-amylopectin chains.
Cellulose-based plastic films were prepared in homogeneous media with a range of lauroyl fatty acid attachments by ester formation, expressed as the degree of substitution (DS). The esters were cast to form films and their mechanical properties studied. This study showed a surprising relationship between DS and elastic modulus as well as DS and tensile strength, where a peak was observed at DS 2.4. The unexpected variation of static elastic modulus (E) was confirmed by dynamic mechanical analysis (DMA) and this trend was also observed for glass transition temperature (Tg). These results are discussed in relation to sample cohesion.
Starch defines a semicrystalline polymer made of two different polysaccharide fractions. The A-and B-type crystalline lattices define the distinct structures reported in cereal and tuber starches, respectively. Amylopectin, the major fraction of starch, is thought to be chiefly responsible for this semicrystalline organization while amylose is generally considered as an amorphous polymer with little or no impact on the overall crystalline organization. STA2 represents a Chlamydomonas reinhardtii gene required for both amylose biosynthesis and the presence of significant granule-bound starch synthase I (GBSSI) activity. We show that this locus encodes a 69 kDa starch synthase and report the organization of the corresponding STA2 locus. This enzyme displays a specific activity an order of magnitude higher than those reported for most vascular plants. This property enables us to report a detailed characterization of amylose synthesis both in vivo and in vitro. We show that GBSSI is capable of synthesizing a significant number of crystalline structures within starch. Quantifications of amount and type of crystals synthesized under these conditions show that GBSSI induces the formation of B-type crystals either in close association with pre-existing amorphous amylopectin or by crystallization of entirely de novo synthesized material.Keywords: starch; amylose synthesis; granule-bound starch synthase; Chlamydomonas reinhardtii; in vitro synthesis.Starch accumulates in plants as a complex granular mixture of a-glucans (a-1,4-linked and a-1,6-branched) consisting chiefly of amylopectin and amylose. In amylopectin, the major fraction is composed of small-size a-1,4-linked chains that are clustered together by the presence of 5% a-1,6 linkages [1] (starch structure reviewed in [2] and [3]; starch metabolism reviewed in [4]). Amylose is composed of longer chains with less than 1% a-1,6 branches. Plant starch can be further distinguished from glycogen by the presence of highly ordered parallel arrays of double helical glucans (reviewed in [5]). The origin of these arrays resides in the close packing of the a-1,6 linkages at the root of the unit amylopectin cluster. The 9 nm size of each repetitive unit or cluster is conserved throughout the plant kingdom [6]. Two major types of crystalline organization have been documented so far in native starch granules. A-type powder diffraction patterns can be recovered from most cereal endosperm and Chlamydomonas reinhardtii starches while B-type structures were reported for tuber starches or high amylose starches from mutants of algae and cereals. It is generally assumed that amylopectin plays a major role in establishing the crystalline organization of starch. Indeed, amylosedefective mutants or antisense constructs of maize and potato accumulate normal amounts of starch with the same A-or B-type granule organization and similar crystallinities to the corresponding wild-type references. In addition, starches with elevated amylose content are generally less crystalline suggesting that ...
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