title: A novel branching enzyme of the GH-57 family in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.
abstract: Branching enzyme (BE) catalyzes formation of the branch points in glycogen and amylopectin by cleavage of the alpha-1,4 linkage and its subsequent transfer to the alpha-1,6 position. We have identified a novel BE encoded by an uncharacterized open reading frame (TK1436) of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. TK1436 encodes a conserved protein showing similarity to members of glycoside hydrolase family 57 (GH-57 family). At the C terminus of the TK1436 protein, two copies of a helix-hairpin-helix (HhH) motif were found. TK1436 orthologs are distributed in archaea of the order Thermococcales, cyanobacteria, some actinobacteria, and a few other bacterial species. When recombinant TK1436 protein was incubated with amylose used as the substrate, a product peak was detected by high-performance anion-exchange chromatography, eluting more slowly than the substrate. Isoamylase treatment of the reaction mixture significantly increased the level of short-chain alpha-glucans, indicating that the reaction product contained many alpha-1,6 branching points. The TK1436 protein showed an optimal pH of 7.0, an optimal temperature of 70 degrees C, and thermostability up to 90 degrees C, as determined by the iodine-staining assay. These properties were the same when a protein devoid of HhH motifs (the TK1436DeltaH protein) was used. The average molecular weight of branched glucan after reaction with the TK1436DeltaH protein was over 100 times larger than that of the starting substrate. These results clearly indicate that TK1436 encodes a structurally novel BE belonging to the GH-57 family. Identification of an overlooked BE species provides new insights into glycogen biosynthesis in microorganisms.
authors.affiliation: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
authors.forename: Taira
authors.initials: T
authors.lastname: Murakami
authors.forename: Tamotsu
authors.initials: T
authors.lastname: Kanai
authors.forename: Hiroki
authors.initials: H
authors.lastname: Takata
authors.forename: Takashi
authors.initials: T
authors.lastname: Kuriki
authors.forename: Tadayuki
authors.initials: T
authors.lastname: Imanaka
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D004789:Enzyme Activation; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D011134:Polysaccharides / Q000494:pharmacology; D011487:Protein Conformation; D011994:Recombinant Proteins; D013696:Temperature; D019712:Thermococcus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins; C008315:maltodextrin