The role of the extra helix alpha zero in the N-terminal extension of the eight-fold beta alpha barrel of indoleglycerol phosphate synthase was probed by point mutation and truncation. Replacing invariant leucine 5 by valine of the enzyme from Escherichia coli affected neither kcat nor Km, 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 kcat value as the wild-type protein but a 220-fold greater Km value. These results suggest that the N-terminal portion of helix alpha zero 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.