Functional characterization of three (GH13) branching enzymes involved in cyanobacterial starch biosynthesis from Cyanobacterium sp. NBRC 102756.
Starch and glycogen are widespread storage polysaccharides in bacteria, plants, and animals. Recently, some cyanobacteria were found to accumulate water-insoluble α-glucan similar to amylopectin rather than glycogen, the latter of which is more commonly produced in these organisms. The amylopectin-producing species including Cyanobacterium sp. NBRC 102756 invariably have three branching enzyme (BE) homologs, BE1, BE2, and BE3, all belonging to the glycoside hydrolase family 13. Multiple BE isoforms in prokaryotes have not been previously studied. In the present work, we carried out functional characterization of these enzymes expressed in Escherichia coli. The recombinant enzymes were all active, although the specific activity of BE3 was much lower than those of BE1 and BE2. After the incubation of the enzymes with amylopectin or amylose, the reaction products were analyzed by fluorophore-assisted carbohydrate capillary electrophoresis method. BE1 and BE2 showed similar chain-length preference to BEIIb isoform of rice (Oryza sativa L.), while the catalytic specificity of BE3 was similar to that of rice BEI. These results indicate that starch-producing cyanobacteria have both type-I BE (BE3) and type-II BEs (BE1 and BE2) in terms of chain-length preferences, as is the case of plants. All BE isoforms were active against phosphorylase limit dextrin, in which outer branches had been uniformly diminished to 4 glucose residues. Based on its catalytic properties, BE3 was assumed to have a role to transfer the glucan chain bearing branch(es) to give rise to a newly growing unit, or cluster as observed in amylopectin molecule.
Department of Biological Production, Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195, Japan. Electronic address: ryuichi@akita-pu.ac.jp.
Ryuichiro
R
Suzuki
Department of Biological Production, Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195, Japan.
Keiichi
K
Koide
Department of Biological Production, Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195, Japan.
Mari
M
Hayashi
Biotechnology Center, Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195, Japan.
Tomoko
T
Suzuki
Biotechnology Center, Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195, Japan.
Takayuki
T
Sawada
Biotechnology Center, Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195, Japan.
Takashi
T
Ohdan
Department of Biological Production, Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195, Japan.
Hidekazu
H
Takahashi
Department of Biological Production, Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195, Japan.
Yasunori
Y
Nakamura
Department of Biological Production, Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195, Japan.
Naoko
N
Fujita
Department of Biological Production, Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195, Japan. Electronic address: esuzuki@akita-pu.ac.jp.
Eiji
E
Suzuki
D015061:1,4-alpha-Glucan Branching Enzyme / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D000595:Amino Acid Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D013213:Starch / Q000096:biosynthesis*
D007527:Isoenzymes; D013213:Starch; D015061:1,4-alpha-Glucan Branching Enzyme
Amylopectin; Branching enzyme; Cyanobacteria; Cyanobacterial starch; Glycogen