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Journal of Bacteriology, August 2006, p. 5915-5924, Vol. 188, No. 16
0021-9193/06/$08.00+0     doi:10.1128/JB.00390-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

A Novel Branching Enzyme of the GH-57 Family in the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

Taira Murakami,1 Tamotsu Kanai,1 Hiroki Takata,2 Takashi Kuriki,2 and Tadayuki Imanaka1*

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan,1 Biochemical Research Laboratory, Ezaki Glico Co., Ltd., Nishiyodogawa-ku, Osaka, 555-8502, Japan2

Received 20 March 2006/ Accepted 29 May 2006

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°C, and thermostability up to 90°C, as determined by the iodine-staining assay. These properties were the same when a protein devoid of HhH motifs (the TK1436{Delta}H protein) was used. The average molecular weight of branched glucan after reaction with the TK1436{Delta}H 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.

* Corresponding author. Mailing address: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan. Phone: 81-75-383-2777. Fax: 81-75-383-2778. E-mail: imanaka{at}sbchem.kyoto-u.ac.jp.

Journal of Bacteriology, August 2006, p. 5915-5924, Vol. 188, No. 16
0021-9193/06/$08.00+0     doi:10.1128/JB.00390-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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