Role of the N-Terminal Extension of the (βα)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.

Functional Identification of the General Acid and Base in the Dehydration Step of Indole-3-glycerol Phosphate Synthase Catalysis

“…Site-directed Mutagenesis of ssIGPS-Initial site-directed mutagenesis of the A/T-rich ssIGPS gene cloned into pET21b (17) proved problematic. To help solve this problem, we obtained an E. coli codon usage-optimized version of ssIGPS (GenScript) that had a richer G/C sequence and cloned it into pET101 using the Champion pET Directional TOPO expression kit (Invitrogen).…”

“…Protein Overexpression, Purification, and CharacterizationThe overexpression and purification of WT and variant ssIGPS enzymes followed protocols described previously (11,17,18). Steady-state enzyme assays for ssIGPS followed previously established procedures (11,19,20).…”

H2rs: Deducing evolutionary and functionally important residue positions by means of an entropy and similarity based analysis of multiple sequence alignments

“…The enzyme indole-3-glycerol phosphate synthase (TrpC) catalyzes the ring closure of an N-alkylated anthranilate to a 3-alkyl indole derivative, which is the fourth step in the tryptophan biosynthesis. It adopts the widespread (βα) 8 -barrel fold and has been studied in detail [59]. Here, we utilized the structure of TrpC from Sulfolobus solfataricus (ssTrpC, PDB ID 1A53) .…”

A novel genetic system for recombinant protein secretion in the Antarctic Pseudoalteromonas haloplanktis TAC125

“…For instance, Ss IGPS displays two exposed loops, located at the N-terminal region, accessible to the action of a host protease [22]. If a single cleavage occurs in this region the activity of truncated enzyme results to be affected [23]. This evidence can justify the absence of Ss IGPS enzymatic activity in the extra-cellular samples of P. haloplanktis TAC125(pFF amyΔ Ct- trpC ) cells.…”