A novel tryptophan synthase beta-subunit from the hyperthermophile Thermotoga maritima. Quaternary structure, steady-state kinetics, and putative physiological role.
Tryptophan synthase catalyzes the last two steps in the biosynthesis of the amino acid tryptophan. The enzyme is an alpha beta beta alpha complex in mesophilic microorganisms. The alpha-subunit (TrpA) catalyzes the cleavage of indoleglycerol phosphate to glyceraldehyde 3-phosphate and indole, which is channeled to the active site of the associated beta-subunit (TrpB1), where it reacts with serine to yield tryptophan. The TrpA and TrpB1 proteins are encoded by the adjacent trpA and trpB1 genes in the trp operon. The genomes of many hyperthermophilic microorganisms, however, contain an additional trpB2 gene located outside of the trp operon. To reveal the properties and potential physiological role of TrpB2, the trpA, trpB1, and trpB2 genes of Thermotoga maritima were expressed heterologously in Escherichia coli, and the resulting proteins were purified and characterized. TrpA and TrpB1 form the familiar alpha beta beta alpha complex, in which the two different subunits strongly activate each other. In contrast, TrpB2 forms a beta(2)-homodimer that has a high catalytic efficiency k(cat)/K(m)(indole) because of a very low K(m)(indole) but does not bind to TrpA. These results suggest that TrpB2 acts as an indole rescue protein, which prevents the escape of this costly hydrophobic metabolite from the cell at the high growth temperatures of hyperthermophiles.
Hettwer
Stefan
S
Universität zu Köln, Institut für Biochemie, Otto-Fischer-Str. 12-14, D-50674 Köln, Germany.
Sterner
Reinhard
R
D000595:Amino Acid Sequence; D002384:Catalysis; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008954:Models, Biological; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology*; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D014367:Tryptophan Synthase / Q000737:chemistry* / Q000502:physiology*
D014364:Tryptophan; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D014367:Tryptophan Synthase