Diversity of reaction characteristics of glucan branching enzymes and the fine structure of α-glucan from various sources.

To investigate the functional properties of 10 α-glucan branching enzymes (BEs) from various sources, we determined the chain-length distribution of BE enzymatic products and their phosphorylase-limit dextrins (Φ-LD). All BEs could be classified into either of the three rice BE isozymes: OsBEI, OsBEIIa, or OsBEIIb. Escherichia coli BE (EcoBE) had the same enzymatic properties as OsBEI, while Synechococcus elongatus BE (ScoBE) and Chlorella kessleri BE (ChlBE) had BEIIb-type properties. Human BE (HosBE), yeast BE (SacBE), and two Porphyridium purpureum BEs (PopBE1 and PopBE2) exhibited the OsBEIIa-type properties. Analysis of chain-length profile of Φ-LD of the BE reaction products revealed that EcoBE, ScoBE, PopBE1, and PopBE2 preferred A-chains as acceptors, while OsBEIIb used B-chains more frequently than A-chains. Both EcoBE and ScoBE specifically formed the branch linkages at the third glucose residue from the reducing end of the acceptor chain. The present results provide evidence for the first time that great variation exists as to the preference of BEs for their acceptor chain, either A-chain or B-chain. In addition, EcoBE and ScoBE recognize the location of branching points in their acceptor chain during their branching reaction. Nevertheless, no correlation exists between the primary structure of BE proteins and their enzymatic characteristics.

The primitive rhodophyte Cyanidioschyzon merolae contains a semiamylopectin-type, but not an amylose-type, alpha-glucan.

The storage glucans of Cyanidioschyzon merolae [clade L-1 (cyanidian algae), order Porphyridiales, subclass Bangiophycidae], which is considered to be one of the most primitive rhodophytes, were analyzed to understand the early evolution of the glucan structure in the Rhodophyta. Chain-length distribution analysis of the glucans of cyanidian algae demonstrated that while the glucans of Cyanidium caldarium and Galdieria sulphuraria are of the glycogen type, those of C. merolae are of the semiamylopectin type, as in other lineages of the Rhodophyta. Gel permeation chromatography, however, showed that the glucans of C. merolae do not include amylose, being different from those of other Bangiophycidae species. Identification by MALDI-TOF-MS and enzyme assaying of glucan granule-bound proteins indicated that phosphorylase, but not starch synthase, is included. Thus, C. merolae has an unusual glucan and bound-protein composition for the Bangiophycidae, appearing to be a member of the Florideophycidae. The finding that the alga does not contain amylose or the related enzyme, granule-bound starch synthase, is, however, consistent with previously reported results of molecular phylogenetic analysis of starch synthases. Our results support an evolutionary scenario defined by the loss of starch and reversion to glycogen synthesis during the evolution of cyanidian algae, and suggest the possibility that a C. merolae-like primitive rhodophyte might have evolved into the Florideophycidae.

Variation in storage alpha-polyglucans of red algae: amylose and semi-amylopectin types in Porphyridium and glycogen type in Cyanidium.

Red algae are widely known to produce floridean starch but it remains unclear whether the molecular structure of this algal polyglucan is distinct from that of the starch synthesized by vascular plants and green algae. The present study shows that the unicellular species Porphyridium purpureum R-1 (order Porphyridiales, class Bangiophyceae) produces both amylopectin-type and amylose-type alpha-polyglucans. In contrast, Cyanidium caldarium (order Porphyridiales, class Bangiophyceae) synthesizes glycogen-type polyglucan, but not amylose. Detailed analysis of alpha-1,4-chain length distribution of P. purpureum polyglucan suggests that the branched polyglucan has a less ordered structure, referred to as semi-amylopectin, as compared with amylopectin of rice endosperm having a tandem-cluster structure. The P. purpureum linear amylose-type polyglucan, which has a lambda(max) of 630 nm typical of amylose-iodine complex and is resistant to Pseudomonas isoamylase digestion, accounts for less than 10% of the total polyglucans. We produced and isolated a cDNA encoding a granule-bound starch synthase (GBSS)-type protein of P. purpureum, which is probably the approximately 60-kDa protein bound tightly to the starch granules, resembling the amylose-synthesizing GBSS protein of green plants. The present investigation indicates that the class Bangiophyceae includes species producing both semi-amylopectin and amylose, and species producing glycogen alone.

Variation in storage alpha-glucans of the Porphyridiales (Rhodophyta).

Storage glucans were analyzed in the Porphyridiales which include the most primitive and phylogenetically diverged species in the Rhodophyta, to understand early evolution of the glucan structure in the Rhodophyta. The storage glucans of both Galdieria sulphuraria and Cyanidium caldarium consisted of glycogen, while those of Rhodosorus marinus, Porphyridium purpureum, P. sordidum and Rhodella violacea could be defined as semi-amylopectin. X-ray diffraction analysis of the glucans demonstrated variation in the crystalline structure: the patterns in P. purpureum and R. violacea were of A- and B-types, respectively, while alpha-glucans of R. marinus and P. sordidum displayed structures with lower crystallinity. Electron microscopic observations indicated that the alpha-glucans of P. sordidum consisted of two kinds of granules; a minor component of more dense granules with crystalline leaflets and a major component of softer ones without crystalline structure. Gel permeation chromatography showed that all the species containing the semi-amylopectin-type glucans also contained amylose, although the relative amounts of this fraction were different depending on the species. Our results are consistent with two distinct evolution scenarios defined either by the independent acquisition of semi-crystalline starch-like structures in the different plant lineages or more probably by the loss of starch and reversion to glycogen synthesis in cyanidian algae growing in hot and acid environments.

Granule-bound starch synthase cDNA in Chlorella kessleri 11 h: cloning and regulation of expression by CO(2) concentration.

The cDNA for the granule-bound starch synthase (GBSS; ADP-glucose-starch glucosyltransferase, EC 2.4.1.21) of Chlorella kessleri 11 h was isolated and characterized. CkGBSS encodes a 609-amino acid polypeptide (65,627 Da) that includes an N-terminal hydrophobic signal peptide of 55 amino acids. The deduced amino acid sequence of the mature CkGBSS polypeptide shares a greater identity (65%) to that of the GBSS protein of Chlamydomonas reinhardtii, than to those of vascular plant species, but does not have the extra-long C-terminal sequence found in C. reinhardtii. When CO(2 )concentration was decreased from 3 to 0.04% (air level) in light, the levels of CkGBSS mRNA, CkGBSS protein, and GBSS activity increased. Under this condition, pyrenoid and pyrenoid starch developed, and the relative amount of amylose in starch increased. These observations suggest that low CO(2) level up-regulates GBSS biosynthesis at the transcriptional level.