Deletion mutagenesis as a test of evolutionary relatedness of indoleglycerol phosphate synthase with other TIM barrel enzymes

The role of the extra helix alpha(0) in the IV-terminal extension of the eight-fold beta alpha barrel of indoleglycerol phosphate synthase,vas probed by point mutation and truncation, Replacing invariant leucine 5 by valine of the enzyme from Escherichia coli affected neither k(cat) nor K-M, but deletion of 8 N-terminal residues decreased solubility strongly, The similarly truncated variant from the hyperthermophile Sulfolobus solfataricus was soluble, and had the same k(cat) value as the wild-type protein but a 220-fold greater K-M value, These results suggest that the N-terminal portion of helix alpha(0) provides for strong binding of the substrate, but is not essential for stabilizing the bound transition state, Thus, three enzymes of tryptophan biosynthesis operate essentially as canonical eight-fold beta alpha barrels, as required for their divergent evolution. (C) 1997 Federation of European Biochemical Societies.