title: Role of the N-terminal extension of the (betaalpha)8-barrel enzyme indole-3-glycerol phosphate synthase for its fold, stability, and catalytic activity.
abstract: Indole-3-glycerol phosphate synthase (IGPS) catalyzes the fifth step in the biosynthesis of tryptophan. It belongs to the large and versatile family of (betaalpha)(8)-barrel enzymes but has an unusual N-terminal extension of about 40 residues. Limited proteolysis with trypsin of IGPS from both Sulfolobus solfataricus (sIGPS) and Thermotoga 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 sIGPSDelta(1-26) and tIGPSDelta(1-25) were produced recombinantly in Escherichia coli, purified, and characterized in comparison to the wild-type enzymes. Both sIGPSDelta(1-26) and tIGPSDelta(1-25) 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. sIGPSDelta(1-26) was crystallized, and its X-ray structure was solved at 2.8 A 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 sIGPSDelta(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 alpha-subunit of tryptophan synthase. These (betaalpha)(8)-barrel enzymes catalyze the reactions preceding and following IGPS in tryptophan biosynthesis but lack an N-terminal extension.
authors.affiliation: Institut für Biophysik und physikalische Biochemie, Universität Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany.
authors.forename: Birgit
authors.initials: B
authors.lastname: Schneider
authors.forename: Thorsten
authors.initials: T
authors.lastname: Knöchel
authors.forename: Beatrice
authors.initials: B
authors.lastname: Darimont
authors.forename: Michael
authors.initials: M
authors.lastname: Hennig
authors.forename: Susanne
authors.initials: S
authors.lastname: Dietrich
authors.forename: Karin
authors.initials: K
authors.lastname: Babinger
authors.forename: Kasper
authors.initials: K
authors.lastname: Kirschner
authors.forename: Reinhard
authors.initials: R
authors.lastname: Sterner
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001704:Biopolymers; D002384:Catalysis; D017931:DNA Primers; D004795:Enzyme Stability; D006868:Hydrolysis; D007209:Indole-3-Glycerol-Phosphate Synthase / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid; D048229:Sulfolobus solfataricus / Q000201:enzymology; D020124:Thermotoga maritima / Q000201:enzymology
chemical_list: D001704:Biopolymers; D017931:DNA Primers; D007209:Indole-3-Glycerol-Phosphate Synthase