Th-trpE and Tk-trpG, the genes that encode the two subunits of anthranilate synthase from the hyperthermophilic archaeon Thermococcus kodakaraensis-KOD1, have been expressed independently in Escherichia coli. The anthranilate synthase complex (Tk-AS complex) was obtained by heat-treatment of the mixture of cell-free extracts containing each recombinant protein, Tk-TrpE (alpha subunit) and Tk-TrpG (beta subunit), at 85 degreesC for 10 min. Further purification of TK-AS complex was carried out by anion-exchange chromatography followed by gel-filtration. Molecular mass estimations from gel-filtration chromatography indicated that Tk-AS complex was a heterodimer (alpha beta). The complex displayed both ammonia- and glutamine-dependent anthranilate synthase activities, and could not utilize asparagine as an ammonia donor. The optimal pH was pH 10.0 and tbe optimal temperature was 85 degreesC in both cases. Mg2+ was necessary for the anthranilate synthase activity. At 75 degreesC, the K-m values of chorismate for ammonia- and glutamine-dependent activities were 13.8 and 3.4 muM, respectively. The K-m value of Mg2+ was 20.5 muM. The K-m values of glutamine and NH4Cl were 88 muM and 5.6 mM,respectively. Although Tk-TrpE displayed 47.6% similarity with TrpE of Salmonella typhimurium, conserved amino acid residues proven to be essential for inhibition of enzyme activity by L-tryptophan were not present in Tk-TrpE. Namely, residues corresponding to Glu39, Met293, and Cys465 in the enzyme from S. typhimurium were replaced by Arg28, Thr221, and Ala384 in Tk-TrpE. Nevertheless, significant inhibition by L-tryptophan was observed, with K-m values of 5.25 and 14 muM for ammonia and glutamine-dependent activities, respectively. The inhibition was competitive with respect to chorismate. The results suggest that the amino acid residues involved in the feedback inhibition by L-tryptophan in the case of Th-AS complex are distinct from previously reported anthranilate synthases.