23] Phosphoribosylanthranilate isomerase and indoleglycerol-phosphate synthase: Tryptophan biosynthetic enzymes from Thermotoga maritima

Abstract

Publisher Summary Phosphoribosylanthranilate isomerase (PRAI) and indoleglycerol-phosphate synthase (IGPS) catalyze the fourth and the fifth steps of tryptophan biosynthesis—namely, an Amadori rearrangement of a ribosylamine and the ring closure to the indole nucleus. In Escherichia coli , PRAI and IGPS are fused to give the monomeric bifunctional polypeptide elGPS-PRAI. The artificially separated monofunctional PRAI and IGPS domains were subsequently produced and characterized, and the reaction mechanisms and folding properties of both enzymes and the PRAI from yeast (yPRAI) were studied, using stopped-flow techniques as well as site-directed mutagenesis. However, many mutational studies, especially on eIGPS, were hampered considerably by the instability and insolubility of the produced mutants. As an alternative source of PRAI and IGPS the stable and monofunctional enzymes tPRAI and tIGPS from the hyperthermophilic bacterium Thermotoga maritima (Tm) were chosen. These turned out to be easy to purify after heterologous expression in E.coli and to crystallize. These studies were also undertaken to understand the structural basis of protein thermostability. This chapter describes the production of these proteins and their purification, as well as their steady-state enzyme kinetics and stability properties.