Architecture, Function, Regulation, and Evolution of α-Glucans Metabolic Enzymes in Prokaryotes.

Bacteria have acquired sophisticated mechanisms for assembling and disassembling polysaccharides of different chemistry. α-d-Glucose homopolysaccharides, so-called α-glucans, are the most widespread polymers in nature being key components of microorganisms. Glycogen functions as an intracellular energy storage while some bacteria also produce extracellular assorted α-glucans. The classical bacterial glycogen metabolic pathway comprises the action of ADP-glucose pyrophosphorylase and glycogen synthase, whereas extracellular α-glucans are mostly related to peripheral enzymes dependent on sucrose. An alternative pathway of glycogen biosynthesis, operating via a maltose 1-phosphate polymerizing enzyme, displays an essential wiring with the trehalose metabolism to interconvert disaccharides into polysaccharides. Furthermore, some bacteria show a connection of intracellular glycogen metabolism with the genesis of extracellular capsular α-glucans, revealing a relationship between the storage and structural function of these compounds. Altogether, the current picture shows that bacteria have evolved an intricate α-glucan metabolism that ultimately relies on the evolution of a specific enzymatic machinery. The structural landscape of these enzymes exposes a limited number of core catalytic folds handling many different chemical reactions. In this Review, we present a rationale to explain how the chemical diversity of α-glucans emerged from these systems, highlighting the underlying structural evolution of the enzymes driving α-glucan bacterial metabolism.

Kre6 (yeast 1,6-ß-transglycosylase) homolog, PhTGS, is essential for ß-glucan synthesis in the haptophyte Pleurochrysis haptonemofera.

Haptophytes synthesize unique ß-glucans containing more ß-1,6-linkages than ß-1,3 linkages, as a storage polysaccharide. To understand the mechanism of the synthesis, we investigated the roles of Kre6 (yeast 1,6-ß-transglycosylase) homologs, PhTGS, in the haptophyte Pleurochrysis haptonemofera. RNAi of PhTGS repressed ß-glucan accumulation and simultaneously induced lipid production, suggesting that PhTGS is involved in ß-glucan synthesis and that the knockdown leads to the alteration of the carbon metabolic flow. PhTGS was expressed more in light, where ß-glucan was actively produced by photosynthesis, than in the dark. The crude extract of E. coli expressing PhKre6 demonstrated its activity to incorporate 14C-UDP-glucose into ß-glucan of P. haptonemofera. These findings suggest that PhTGS functions in storage ß-glucan synthesis specifically in light, probably by producing the ß-1,6-branch.

One of the isoamylase isoforms, CMI294C, is required for semi-amylopectin synthesis in the rhodophyte Cyanidioschyzon merolae.

Most rhodophytes synthesize semi-amylopectin as a storage polysaccharide, whereas some species in the most primitive class (Cyanidiophyceae) make glycogen. To know the roles of isoamylases in semi-amylopectin synthesis, we investigated the effects of isoamylase gene (CMI294C and CMS197C)-deficiencies on semi-amylopectin molecular structure and starch granule morphology in Cyanidioschyzon merolae (Cyanidiophyceae). Semi-amylopectin content in a CMS197C-disruption mutant (ΔCMS197C) was not significantly different from that in the control strain, while that in a CMI294C-disruption mutant (ΔCMI294C) was much lower than those in the control strain, suggesting that CMI294C is essential for semi-amylopectin synthesis. Scanning electron microscopy showed that the ΔCMI294C strain contained smaller starch granules, while the ΔCMS197C strain had normal size, but donut-shaped granules, unlike those of the control strain. Although the chain length distribution of starch from the control strain displayed a semi-amylopectin pattern with a peak around degree of polymerization (DP) 11-13, differences in chain length profiles revealed that the ΔCMS197C strain has more short chains (DP of 3 and 4) than the control strain, while the ΔCMI294C strain has more long chains (DP ≥12). These findings suggest that CMI294C-type isoamylase, which can debranch a wide range of chains, probably plays an important role in semi-amylopectin synthesis unique in the Rhodophyta.

Determination of Glucan Chain Length Distribution of Glycogen Using the Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) Method.

Glycogen particles are branched polysaccharides composed of linear chains of glucosyl units linked by α-1,4 glucoside bonds. The latter are attached to each other by α-1,6 glucoside linkages, referred to as branch points. Among the different forms of carbon storage (i.e., starch, ß-glucan), glycogen is probably one of the oldest and most successful storage polysaccharides found across the living world. Glucan chains are organized so that a large amount of glucose can quickly be stored or fueled in a cell when needed. Numerous complementary techniques have been developed over the last decades to solve the fine structure of glycogen particles. This article describes Fluorophore-Assisted Carbohydrate Electrophoresis (FACE). This method quantifies the population of glucan chains that compose a glycogen particle. Also known as chain length distribution (CLD), this parameter mirrors the particle size and the percentage of branching. It is also an essential requirement for the mathematical modeling of glycogen biosynthesis.

Conservation of the glycogen metabolism pathway underlines a pivotal function of storage polysaccharides in Chlamydiae.

The order Chlamydiales includes obligate intracellular pathogens capable of infecting mammals, fishes and amoeba. Unlike other intracellular bacteria for which intracellular adaptation led to the loss of glycogen metabolism pathway, all chlamydial families maintained the nucleotide-sugar dependent glycogen metabolism pathway i.e. the GlgC-pathway with the notable exception of both Criblamydiaceae and Waddliaceae families. Through detailed genome analysis and biochemical investigations, we have shown that genome rearrangement events have resulted in a defective GlgC-pathway and more importantly we have evidenced a distinct trehalose-dependent GlgE-pathway in both Criblamydiaceae and Waddliaceae families. Altogether, this study strongly indicates that the glycogen metabolism is retained in all Chlamydiales without exception, highlighting the pivotal function of storage polysaccharides, which has been underestimated to date. We propose that glycogen degradation is a mandatory process for fueling essential metabolic pathways that ensure the survival and virulence of extracellular forms i.e. elementary bodies of Chlamydiales.

Retracing Storage Polysaccharide Evolution in Stramenopila.

Eukaryotes most often synthesize storage polysaccharides in the cytosol or vacuoles in the form of either alpha (glycogen/starch)- or beta-glucosidic (chrysolaminarins and paramylon) linked glucan polymers. In both cases, the glucose can be packed either in water-soluble (glycogen and chrysolaminarins) or solid crystalline (starch and paramylon) forms with different impacts, respectively, on the osmotic pressure, the glucose accessibility, and the amounts stored. Glycogen or starch accumulation appears universal in all free-living unikonts (metazoa, fungi, amoebozoa, etc.), as well as Archaeplastida and alveolata, while other lineages offer a more complex picture featuring both alpha- and beta-glucan accumulators. We now infer the distribution of these polymers in stramenopiles through the bioinformatic detection of their suspected metabolic pathways. Detailed phylogenetic analysis of key enzymes of these pathways correlated to the phylogeny of Stramenopila enables us to retrace the evolution of storage polysaccharide metabolism in this diverse group of organisms. The possible ancestral nature of glycogen metabolism in eukaryotes and the underlying source of its replacement by beta-glucans are discussed.

Analyses of 202 plastid genomes elucidate the phylogeny of Solanum section Petota.

Our paper analyzes full plastid DNA sequence data of 202 wild and cultivated diploid potatoes, Solanum section Petota, to explore its phylogenetic utility compared to prior analyses of the same accessions using genome-wide nuclear SNPs, and plastid DNA restriction site data. The present plastid analysis discovered the same major clades as the nuclear data but with some substantial differences in topology within the clades. The considerably larger plastid and nuclear data sets add phylogenetic resolution within the prior plastid DNA restriction site data, highlight plastid/nuclear incongruence that supports hypotheses of hybridization/introgression to help explain the taxonomic difficulty in the section.

Genomic Analyses Yield Markers for Identifying Agronomically Important Genes in Potato.

Wild potato species have substantial phenotypic and physiological diversity. Here, we report a comprehensive assessment of wild and cultivated potato species based on genomic analyses of 201 accessions of Solanum section Petota. We sequenced the genomes of these 201 accessions and identified 6 487 006 high-quality single nucleotide polymorphisms (SNPs) from 167 accessions in clade 4 of Solanum section Petota, including 146 wild and 21 cultivated diploid potato accessions with a broad geographic distribution. Genome-wide genetic variation analysis showed that the diversity of wild potatoes is higher than that of cultivated potatoes, and much higher genetic diversity in the agronomically important disease resistance genes was observed in wild potatoes. Furthermore, by exploiting information about known quantitative trait loci (QTL), we identified 609 genes under selection, including those correlated with the loss of bitterness in tubers and those involved in tuberization, two major domesticated traits of potato. Phylogenetic analyses revealed a north-south division of all species in clade 4, not just those in the S. brevicaule complex, and further supported S. candolleanum as the progenitor of cultivated potato and the monophyletic origin of cultivated potato in southern Peru. In addition, we analyzed the genome of S. candolleanum and identified 529 genes lost in cultivated potato. Collectively, the molecular markers generated in this study provide a valuable resource for the identification of agronomically important genes useful for potato breeding.

Bound Substrate in the Structure of Cyanobacterial Branching Enzyme Supports a New Mechanistic Model.

Branching enzyme (BE) catalyzes the formation of α-1,6-glucosidic linkages in amylopectin and glycogen. The reaction products are variable, depending on the organism sources, and the mechanistic basis for these different outcomes is unclear. Although most cyanobacteria have only one BE isoform belonging to glycoside hydrolase family 13, Cyanothece sp. ATCC 51142 has three isoforms (BE1, BE2, and BE3) with distinct enzymatic properties, suggesting that investigations of these enzymes might provide unique insights into this system. Here, we report the crystal structure of ligand-free wild-type BE1 (residues 5-759 of 1-773) at 1.85 Å resolution. The enzyme consists of four domains, including domain N, carbohydrate-binding module family 48 (CBM48), domain A containing the catalytic site, and domain C. The central domain A displays a (ß/α)8-barrel fold, whereas the other domains adopt ß-sandwich folds. Domain N was found in a new location at the back of the protein, forming hydrogen bonds and hydrophobic interactions with CBM48 and domain A. Site-directed mutational analysis identified a mutant (W610N) that bound maltoheptaose with sufficient affinity to enable structure determination at 2.30 Å resolution. In this structure, maltoheptaose was bound in the active site cleft, allowing us to assign subsites -7 to -1. Moreover, seven oligosaccharide-binding sites were identified on the protein surface, and we postulated that two of these in domain A served as the entrance and exit of the donor/acceptor glucan chains, respectively. Based on these structures, we propose a substrate binding model explaining the mechanism of glycosylation/deglycosylation reactions catalyzed by BE.

Biotic Host-Pathogen Interactions As Major Drivers of Plastid Endosymbiosis.

The plastid originated 1.5 billion years ago through a primary endosymbiosis involving a heterotrophic eukaryote and an ancient cyanobacterium. Phylogenetic and biochemical evidence suggests that the incipient endosymbiont interacted with an obligate intracellular chlamydial pathogen that housed it in an inclusion. This aspect of the ménage-à-trois hypothesis (MATH) posits that Chlamydiales provided critical novel transporters and enzymes secreted by the pathogens in the host cytosol. This initiated the efflux of photosynthate to both the inclusion lumen and host cytosol. Here we review the experimental evidence supporting the MATH and focus on chlamydial genes that replaced existing cyanobacterial functions. The picture emerging from these studies underlines the importance of chlamydial host-pathogen interactions in the metabolic integration of the primary plastid.

Was the Chlamydial Adaptative Strategy to Tryptophan Starvation an Early Determinant of Plastid Endosymbiosis?

Chlamydiales were recently proposed to have sheltered the future cyanobacterial ancestor of plastids in a common inclusion. The intracellular pathogens are thought to have donated those critical transporters that triggered the efflux of photosynthetic carbon and the consequent onset of symbiosis. Chlamydiales are also suspected to have encoded glycogen metabolism TTS (Type Three Secretion) effectors responsible for photosynthetic carbon assimilation in the eukaryotic cytosol. We now review the reasons underlying other chlamydial lateral gene transfers evidenced in the descendants of plastid endosymbiosis. In particular we show that half of the genes encoding enzymes of tryptophan synthesis in Archaeplastida are of chlamydial origin. Tryptophan concentration is an essential cue triggering two alternative modes of replication in Chlamydiales. In addition, sophisticated tryptophan starvation mechanisms are known to act as antibacterial defenses in animal hosts. We propose that Chlamydiales have donated their tryptophan operon to the emerging plastid to ensure increased synthesis of tryptophan by the plastid ancestor. This would have allowed massive expression of the tryptophan rich chlamydial transporters responsible for symbiosis. It would also have allowed possible export of this valuable amino-acid in the inclusion of the tryptophan hungry pathogens. Free-living single cell cyanobacteria are devoid of proteins able to transport this amino-acid. We therefore investigated the phylogeny of the Tyr/Trp transporters homologous to E. coli TyrP/Mre and found yet another LGT from Chlamydiales to Archaeplastida thereby considerably strengthening our proposal.

Comparison of Chain-Length Preferences and Glucan Specificities of Isoamylase-Type α-Glucan Debranching Enzymes from Rice, Cyanobacteria, and Bacteria.

It has been believed that isoamylase (ISA)-type α-glucan debranching enzymes (DBEs) play crucial roles not only in α-glucan degradation but also in the biosynthesis by affecting the structure of glucans, although molecular basis on distinct roles of the individual DBEs has not fully understood. In an attempt to relate the roles of DBEs to their chain-length specificities, we analyzed the chain-length distribution of DBE enzymatic reaction products by using purified DBEs from various sources including rice, cyanobacteria, and bacteria. When DBEs were incubated with phytoglycogen, their chain-length specificities were divided into three groups. First, rice endosperm ISA3 (OsISA3) and Eschericia coli GlgX (EcoGlgX) almost exclusively debranched chains having degree of polymerization (DP) of 3 and 4. Second, OsISA1, Pseudomonas amyloderamosa ISA (PsaISA), and rice pullulanase (OsPUL) could debranch a wide range of chains of DP≧3. Third, both cyanobacteria ISAs, Cyanothece ATCC 51142 ISA (CytISA) and Synechococcus elongatus PCC7942 ISA (ScoISA), showed the intermediate chain-length preference, because they removed chains of mainly DP3-4 and DP3-6, respectively, while they could also react to chains of DP5-10 and 7-13 to some extent, respectively. In contrast, all these ISAs were reactive to various chains when incubated with amylopectin. In addition to a great variation in chain-length preferences among various ISAs, their activities greatly differed depending on a variety of glucans. Most strikingly, cyannobacteria ISAs could attack branch points of pullulan to a lesser extent although no such activity was found in OsISA1, OsISA3, EcoGlgX, and PsaISA. Thus, the present study shows the high possibility that varied chain-length specificities of ISA-type DBEs among sources and isozymes are responsible for their distinct functions in glucan metabolism.

Characterization of Function of the GlgA2 Glycogen/Starch Synthase in Cyanobacterium sp. Clg1 Highlights Convergent Evolution of Glycogen Metabolism into Starch Granule Aggregation.

At variance with the starch-accumulating plants and most of the glycogen-accumulating cyanobacteria, Cyanobacterium sp. CLg1 synthesizes both glycogen and starch. We now report the selection of a starchless mutant of this cyanobacterium that retains wild-type amounts of glycogen. Unlike other mutants of this type found in plants and cyanobacteria, this mutant proved to be selectively defective for one of the two types of glycogen/starch synthase: GlgA2. This enzyme is phylogenetically related to the previously reported SSIII/SSIV starch synthase that is thought to be involved in starch granule seeding in plants. This suggests that, in addition to the selective polysaccharide debranching demonstrated to be responsible for starch rather than glycogen synthesis, the nature and properties of the elongation enzyme define a novel determinant of starch versus glycogen accumulation. We show that the phylogenies of GlgA2 and of 16S ribosomal RNA display significant congruence. This suggests that this enzyme evolved together with cyanobacteria when they diversified over 2 billion years ago. However, cyanobacteria can be ruled out as direct progenitors of the SSIII/SSIV ancestral gene found in Archaeplastida. Hence, both cyanobacteria and plants recruited similar enzymes independently to perform analogous tasks, further emphasizing the importance of convergent evolution in the appearance of starch from a preexisting glycogen metabolism network.

Crystallization and crystallographic analysis of branching enzymes from Cyanothece sp. ATCC 51142.

Several cyanobacterial species, including Cyanothece sp. ATCC 51142, remarkably have four isoforms of α-glucan branching enzymes (BEs). Based on their primary structures, they are classified into glycoside hydrolase (GH) family 13 (BE1, BE2 and BE3) or family 57 (GH57 BE). In the present study, GH13-type BEs from Cyanothece sp. ATCC 51142 (BE1, BE2 and BE3) have been overexpressed in Escherichia coli and biochemically characterized. The recombinant BE1 was crystallized by the hanging-drop vapour-diffusion method. Crystals of BE1 were obtained at 293 K in the presence of 0.2 M Mg(2+), 7-10%(w/v) ethanol, 0.1 M HEPES-NaOH pH 7.2-7.9. The crystals belonged to the tetragonal space group P41212, with unit-cell parameters a = b = 133.75, c = 185.90 Å, and diffracted to beyond 1.85 Šresolution. Matthews coefficient calculations suggested that the crystals of BE1 contained two molecules in the asymmetric unit.

Toward an understanding of the function of Chlamydiales in plastid endosymbiosis.

Plastid endosymbiosis defines a process through which a fully evolved cyanobacterial ancestor has transmitted to a eukaryotic phagotroph the hundreds of genes required to perform oxygenic photosynthesis, together with the membrane structures, and cellular compartment associated with this process. In this review, we will summarize the evidence pointing to an active role of Chlamydiales in metabolic integration of free living cyanobacteria, within the cytosol of the last common plant ancestor.

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.

Transition from glycogen to starch metabolism in Archaeplastida.

In this opinion article we propose a scenario detailing how two crucial components have evolved simultaneously to ensure the transition of glycogen to starch in the cytosol of the Archaeplastida last common ancestor: (i) the recruitment of an enzyme from intracellular Chlamydiae pathogens to facilitate crystallization of α-glucan chains; and (ii) the evolution of novel types of polysaccharide (de)phosphorylating enzymes from preexisting glycogen (de)phosphorylation host pathways to allow the turnover of such crystals. We speculate that the transition to starch benefitted Archaeplastida in three ways: more carbon could be packed into osmotically inert material; the host could resume control of carbon assimilation from the chlamydial pathogen that triggered plastid endosymbiosis; and cyanobacterial photosynthate export could be integrated in the emerging Archaeplastida.

Convergent evolution of polysaccharide debranching defines a common mechanism for starch accumulation in cyanobacteria and plants.

Starch, unlike hydrosoluble glycogen particles, aggregates into insoluble, semicrystalline granules. In photosynthetic eukaryotes, the transition to starch accumulation occurred after plastid endosymbiosis from a preexisting cytosolic host glycogen metabolism network. This involved the recruitment of a debranching enzyme of chlamydial pathogen origin. The latter is thought to be responsible for removing misplaced branches that would otherwise yield a water-soluble polysaccharide. We now report the implication of starch debranching enzyme in the aggregation of semicrystalline granules of single-cell cyanobacteria that accumulate both glycogen and starch-like polymers. We show that an enzyme of analogous nature to the plant debranching enzyme but of a different bacterial origin was recruited for the same purpose in these organisms. Remarkably, both the plant and cyanobacterial enzymes have evolved through convergent evolution, showing novel yet identical substrate specificities from a preexisting enzyme that originally displayed the much narrower substrate preferences required for glycogen catabolism.

Chlamydia, cyanobiont, or host: who was on top in the ménage à trois?

The endosymbiont hypothesis proposes that photosynthate from the cyanobiont was exported to the cytosol of the eukaryote host and polymerized from ADP-glucose into glycogen. Chlamydia-like pathogens are the second major source of foreign genes in Archaeplastida, suggesting that these obligate intracellular pathogens had a significant role during the establishment of endosymbiosis, likely through facilitating the metabolic integration between the endosymbiont and the eukaryotic host. In this opinion article, we propose that a hexose phosphate transporter of chlamydial origin was the first transporter responsible for exporting photosynthate out of the cyanobiont. This connection pre-dates the recruitment of the host-derived carbon translocators on the plastid inner membranes of green and red algae, land plants, and photosynthetic organisms of higher order endosymbiotic origin.