Functional characterization of three (GH13) branching enzymes involved in cyanobacterial starch biosynthesis from Cyanobacterium sp. NBRC 102756

Suzuki, Ryuichiro; Koide, Keiichi; Hayashi, Makio; Suzuki, Tomoko; Sawada, Takayuki; Ohdan, Takashi; Takahashi, Hidekazu; Nakamura, Yasunori; Fujita, Naoko; Suzuki, Eiji

doi:10.1016/j.bbapap.2015.02.012

Cited by 24 publications

(18 citation statements)

References 52 publications

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“…These groups were based on the fact that BE2 and BE3 lacked either 97 or 111 amino acids at the N terminus, consistent with observations made by others (10,12), and that there were various insertions and deletions in the catalytic A-domain region (see Fig. S2) (14). Some regular glycogen-forming species, such as Synechocystis sp.…”

Section: Resultssupporting

confidence: 54%

“…Analysis of storage polysaccharides from Cyanothece sp. ATCC 51142, Cyanobacterium strain Clg1, and Cyanobacterium strain NBRC 102756 revealed that their storage granules have a molecular mass virtually indistinguishable from that of amylopectin (9,14). Moreover, the thermal properties, crystallinity, and branching structure are similar to those of amylopectin, and the semi-amylopectin material synthesized by these strains is organized in tandem cluster structures.…”

mentioning

confidence: 76%

“…Additional alignment of the N-terminal and catalytic domain regions of the branching enzymes was performed similarly with ClustalO. The alignment file was then imported into ESPript 3 (http://espript.ibcp.fr/ESPript/ESPript/), and secondarystructure features were rendered from using PDB Chain ID 1M7X as a reference (http://www.rcsb.org/pdb), similar to analysis performed by Suzuki et al (14).…”

Section: Methodsmentioning

confidence: 99%

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Altering the Structure of Carbohydrate Storage Granules in the Cyanobacterium Synechocystis sp. Strain PCC 6803 through Branching-Enzyme Truncations

2016

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Carbohydrate storage is an important element of metabolism in cyanobacteria and in the chloroplasts of plants. Understanding how to manipulate the metabolism and storage of carbohydrate is also an important factor toward harnessing cyanobacteria for energy production. While most cyanobacteria produce glycogen, some have been found to accumulate polysaccharides in the form of water-insoluble ␣-glucan similar to amylopectin. Notably, this alternative form, termed "semi-amylopectin," forms in cyanobacterial species harboring three branching-enzyme (BE) homologs, designated BE1, BE2, and BE3. In this study, mutagenesis of the branching genes found in Synechocystis sp. strain PCC 6803 was performed in order to characterize their possible impact on polysaccharide storage granule morphology. N-terminal truncations were made to the native BE gene of Synechocystis sp. PCC 6803. In addition, one of the two native debranching enzyme genes was replaced with a heterologous debranching enzyme gene from a semi-amylopectin-forming strain. Growth and glycogen content of mutant strains did not significantly differ from those of the wild type, and ultrastructure analysis revealed only slight changes to granule morphology. However, analysis of chain length distribution by anion-exchange chromatography revealed modest changes to the branchedchain length profile. The resulting glycogen shared structure characteristics similar to that of granules isolated from semi-amylopectin-producing strains. IMPORTANCEThis study is the first to investigate the impact of branching-enzyme truncations on the structure of storage carbohydrates in cyanobacteria. The results of this study are an important contribution toward understanding the relationship between the enzymatic repertoire of a cyanobacterial species and the morphology of its storage carbohydrates. P hotosynthetic carbon assimilation and subsequent storage of generated carbohydrates constitute a crucial process in cyanobacterial cells during diurnal growth. This process occurs in close proximity to the thylakoid membranes that are typically arranged around the central cytoplasmic space or project radially out from the cell poles (1). Most bacteria, including cyanobacteria, produce a form of soluble glycogen (2-4). In fact, the use of glycogen as the polysaccharide storage polymer is widespread in nature and can be found in bacterial, archaeal, and eukaryotic species (5). Glycogen biosynthesis occurs through the sequential actions of ADPglucose pyrophosphorylase (GlgC) glycogen synthase (GlgA) and the branching enzyme (BE; GlgB) (Fig. 1). Briefly, glucose is activated via addition of ADP to become ADP-glucose, which then gets polymerized to the nonreducing end of an ␣-1,4-linked glucan chain. The growing glucose polymer chains are then linked together in the ␣-1,6 position by the branching enzyme (GlgB) via a hydrolytic cleavage reaction whereby an ␣-1,4 linkage is broken and inter-or intramolecular transfer reconnects the chain. These actions continue successively, eventually form...

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Section: Resultssupporting

confidence: 54%

mentioning

confidence: 76%

Section: Methodsmentioning

confidence: 99%

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Altering the Structure of Carbohydrate Storage Granules in the Cyanobacterium Synechocystis sp. Strain PCC 6803 through Branching-Enzyme Truncations

2016

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“…By contrast, ATCC 51142 and NBRC 102756 contain two extra GH13_9 subfamily isoforms (2,16). The three GH13_9 BE isoforms from ATCC 51142 and NBRC 102756 have been characterized, and it was found that BE1 and BE2 transferred short glucan chains with a degree of polymerization (DP) of 6 -7, whereas BE3 transferred short as well as long glucan chains (DP 30) (17,18).…”

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confidence: 99%

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

Hayashi

Suzuki

Colleoni

et al. 2017

Journal of Biological Chemistry

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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, 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 (β/α)-barrel fold, whereas the other domains adopt β-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.

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“…For negatively charged analyte, the electroosmotic flow is entirely suppressed. During the period of this review, size based separations utilizing capillary electrophoresis were almost exclusively used for the analysis of the products of enzymes, such as polymers [47] and DNA [48]. …”

Section: Adapting the Separation To Determine Km Valuesmentioning

confidence: 99%

Advances in enzyme substrate analysis with capillary electrophoresis

Gattu

Crihfield

et al. 2018

Methods

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Capillary electrophoresis provides a rapid, cost-effective platform for enzyme and substrate characterization. The high resolution achievable by capillary electrophoresis enables the analysis of substrates and products that are indistinguishable by spectroscopic techniques alone, while the small volume requirement enables analysis of enzymes or substrates in limited supply. Furthermore, the compatibility of capillary electrophoresis with various detectors makes it suitable for KM determinations ranging from nanomolar to millimolar concentrations. Capillary electrophoresis fundamentals are discussed with an emphasis on the separation mechanisms relevant to evaluate sets of substrate and product that are charged, neutral, and even chiral. The basic principles of Michaelis-Menten determinations are reviewed and the process of translating capillary electrophoresis electropherograms into a Michaelis-Menten curve is outlined. The conditions that must be optimized in order to couple off-line and on-line enzyme reactions with capillary electrophoresis separations, such as incubation time, buffer pH and ionic strength, and temperature, are examined to provide insight into how the techniques can be best utilized. The application of capillary electrophoresis to quantify enzyme inhibition, in the form of KI or IC50 is detailed. The concept and implementation of the immobilized enzyme reactor is described as a means to increase enzyme stability and reusability, as well as a powerful tool for screening enzyme substrates and inhibitors. Emerging techniques focused on applying capillary electrophoresis as a rapid assay to obtain structural identification or sequence information about a substrate and in-line digestions of peptides and proteins coupled to mass spectrometry analyses are highlighted.

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Functional characterization of three (GH13) branching enzymes involved in cyanobacterial starch biosynthesis from Cyanobacterium sp. NBRC 102756

Cited by 24 publications

References 52 publications

Altering the Structure of Carbohydrate Storage Granules in the Cyanobacterium Synechocystis sp. Strain PCC 6803 through Branching-Enzyme Truncations

Altering the Structure of Carbohydrate Storage Granules in the Cyanobacterium Synechocystis sp. Strain PCC 6803 through Branching-Enzyme Truncations

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

Advances in enzyme substrate analysis with capillary electrophoresis

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