Anthranilate synthase without an LLES motif from a hyperthermophilic archaeon is inhibited by tryptophan.
Tk-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 degrees C 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 (alphabeta). 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 the optimal temperature was 85 degrees C in both cases. Mg2+ was necessary for the anthranilate synthase activity. At 75 degrees C, the K(m) values of chorismate for ammonia- and glutamine-dependent activities were 13.8 and 3.4 microM, respectively. The K(m) value of Mg2+ was 20.5 microM. The K(m) values of glutamine and NH4Cl were 88 microM 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(i) values of 5.25 and 74 microM 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 Tk-AS complex are distinct from previously reported anthranilate synthases.
Department of Synthetic Chemistry and Biological Chemistry, Kyoto University, Kyoto, Yoshida-Honmachi, Sakyo-ku, 606-8501, Japan.
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Tang
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Ezaki
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Atomi
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Imanaka
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000643:Ammonium Chloride / Q000494:pharmacology; D000878:Anthranilate Synthase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D002827:Chorismic Acid / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D019766:Genes, Archaeal / Q000235:genetics; D005973:Glutamine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics; D014364:Tryptophan / Q000494:pharmacology*
D000643:Ammonium Chloride; D005973:Glutamine; D014364:Tryptophan; D000878:Anthranilate Synthase; D002827:Chorismic Acid