Role of the N-Terminal Extension of the (βα)₈-Barrel Enzyme Indole-3-glycerol Phosphate Synthase for Its Fold, Stability, and Catalytic Activity^†,‡

Indole-3-glycerol phosphate synthase (IGPS) catalyzes the fifth step in the biosynthesis of tryptophan. It belongs to the large and versatile family of (beta alpha)(8)-barrel enzymes but has an unusual N-terminal extension of about 40 residues. Limited proteolysis with trypsin of IGPS from both Sufolobus solfataricus (sIGPS) and Thermologa maritima (tIGPS) removes about 25 N-terminal residues and one of the two extra helices contained therein. To assess the role of the extension, the N-terminally truncated variants sIGPS Delta(1-26) and tlGPS Delta(1-25) were produced reconibinantly in Escherichia coli, purified, and characterized in comparison to the wild-type enzymes. Both sIGPS Delta(1-26) and tlGPS Delta(I -25) have unchanged oligomerization states and turnover numbers. In contrast, their Michaelis constants for the substrate 1-(O-carboxyphenylamino)-1-deoxyribulose 5-phosphate are increased, and their resistance toward unfolding induced by heat and guanidinium chloride is decreased. sIGPS Delta(1-26) was crystallized, and its X-ray structure was solved at 2.8 angstrom resolution. The comparison with the known structure of sIGPS reveals small differences that account for its reduced substrate affinity and protein stability. The structure of the core of sIGPS Delta(1-26) is, however, unchanged compared to sIGPS, explaining its retained catalytic activity and consistent with the idea that it evolved from the same ancestor as the phosphoribosyl anthranilate isomerase and the a.-subunit of tryptophan synthase. These (beta alpha)(8)-barrel enzymes catalyze the reactions preceding and following IGPS in tryptoplian biosynthesis but lack an N-terminal extension.