Crystal Structure and Functional Characterization of the Bifunctional N-(5'-Phosphoribosyl)anthranilate Isomerase-indole-3-glycerol-phosphate Synthase from Corynebacterium glutamicum.

L-Tryptophan is known as an aromatic amino acid and one of the essential amino acids that must be ingested through various additives or food. TrpCF is a bifunctional enzyme that has indole-glycerol-phosphate synthase (IGPS) and phosphoribosylanthranilate isomerase (PRAI) activity. In this report, we identified the crystal structure of TrpCF from Corynebacterium glutamicum (CgTrpCF) and successfully elucidated the active site by attaching rCdRP similar to the substrate and product of the TrpCF reaction. Also, we revealed that CgTrpCF shows a conformational change at the loops upon substrate binding. We analyzed amino acid sequences of the homologues of CgTrpCF, and the residues of the substrate-binding site in TrpCF were highly conserved except for some residues. These less conserved residues were replaced by site-directed mutagenesis experiments. Consequently, we obtained the CgTrpCFP294K (PRAICD/P294K) variant that has enhanced activity.