Characterization of an Archaeal Cyclodextrin Glucanotransferase with a Novel C-Terminal Domain

Abstract

A gene encoding a cyclodextrin glucanotransferase (CGTase) from Thermococcus kodakaraensis KOD1 (CGT Tk ) was identified and characterized. The gene (cgt Tk ) encoded a protein of 713 amino acid residues harboring the four conserved regions found in all members of the ␣-amylase family. However, the C-terminal domain corresponding to domain E of previously known CGTases displayed a completely distinct primary structure. In order to elucidate the catalytic function of the gene product, the recombinant enzyme was purified by anion-exchange chromatography, and its enzymatic properties were investigated. The enzyme displayed significant starch-degrading activity (750 U/mg of protein) with an optimal temperature and pH of 80°C and 5.5 to 6.0, respectively. The presence of Ca 2؉ enhanced the enzyme activity and elevated the optimum temperature to 85 to 90°C. With the addition of Ca 2؉ , the enzyme showed extreme thermostability, with almost no loss of enzymatic activity after 80 min at 85°C, and a half-life of 20 min at 100°C. CGT Tk could hydrolyze soluble starch and glycogen but failed to hydrolyze pullulan. Most importantly, although CGT Tk harbored a unique C-terminal domain, we found that the protein also exhibited significant CGTase activity, with ␤-cyclodextrin as the main product. In order to identify the involvement, if any, of the C-terminal region in the CGTase activity, we analyzed a truncated protein (CGT Tk ⌬C) with 23 C-terminal amino acid residues deleted. CGT Tk ⌬C displayed similar properties in terms of starch-binding activity, substrate specificity, and thermostability, but unexpectedly showed higher starch-degrading activity than the parental CGT Tk . In contrast, the cyclization activity of CGT Tk ⌬C was abolished. The results indicate that the presence of the structurally novel C-terminal domain is essential for CGT Tk to properly catalyze the cyclization reaction.

Transcriptional and Biochemical Analysis of Starch Metabolism in the Hyperthermophilic Archaeon Pyrococcus furiosus

“…However, it is more similar to proteins that hydrolyze linear polysaccharides and synthesize cyclic forms. Specifically, PF0478 shows 64 and 39% sequence identity with the extracellular cyclomaltodextrin glucanotransferases (CGTases) of Thermococcus kodakaraensis KOD1 (35) and Thermococcus sp. strain B1001 (19), respectively.…”

Biochemical Properties of a Putative Signal Peptide Peptidase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

“…Some representative signal sequences, those from four proteins that have been experimentally proven to be secreted from T. kodakaraensis (19,30,35,36; unpublished data), are shown in Fig. 7.…”

Description of Thermococcus kodakaraensis sp. nov., a well studied hyperthermophilic archaeon previously reported as Pyrococcus sp. KOD1

“…NOV. 265 Consistent with the observed growth on starch and maltooligosaccharides, we found that strain KOD1 harbors a variety of genes involved in polysaccharide degradation, including 4-α-glucanotransferase, α-amylase and cyclodextrin glucanotransferase. The enzyme activities of the respective protein products have been biochemically characterized (Tachibana et al 1996, 1997, Rashid et al 2002a. Strain KOD1 grows well under gluconeogenic conditions with pyruvate and amino acids, and the key enzyme of gluconeogenesis, fructose 1,6-bisphosphatase (FBPase), has been examined.…”