Anthranilate synthase without an LLES motif from a hyperthermophilic archaeon is inhibited by tryptophan

Th-trpE and Tk-trpG, the genes that encode the two subunits of anthranilate synthase from the hyperthermophilic archaeon Thermococcus kodakaraensis-KO D1, have been expressed independently in Escherichia coli. The anthranilate synthase complex (Tk-AS complex) was obtained by heat-treatment of the mix ture of cell-free extracts containing each recombinant protein, Tk-TrpE (al pha subunit) and Tk-TrpG (beta subunit), at 85 degreesC for 10 min. Further purification of TK-AS complex was carried out by anion-exchange chromatogr aphy followed by gel-filtration. Molecular mass estimations from gel-filtra tion chromatography indicated that Tk-AS complex was a heterodimer (alpha b eta). The complex displayed both ammonia- and glutamine-dependent anthranil ate synthase activities, and could not utilize asparagine as an ammonia don or. 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. A t 75 degreesC, the K-m values of chorismate for ammonia- and glutamine-depe ndent 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 Sal monella typhimurium, conserved amino acid residues proven to be essential f or inhibition of enzyme activity by L-tryptophan were not present in Tk-Trp E. Namely, residues corresponding to Glu39, Met293, and Cys465 in the enzym e 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 i nhibition by L-tryptophan in the case of Th-AS complex are distinct from pr eviously reported anthranilate synthases. (C) 2001 Academic Press.