Importance of the MbtH-like protein TioT for production and activation of the thiocoraline adenylation domain of TioK

The 3-hydroxyquinaldic acid (3HQA) chromophores of thiocoraline are essential for the biological DNA bisintercalating function of this antitumor agent. The 3HQA units are also proposed to play a critical role in the resistance mechanism of the thiocoraline-producing organism against this natural product. Because of their important functions, there is a great interest in understanding the 3HQA formation from l-Trp. The first proposed committed steps during 3HQA biosynthesis consist of conversion of l-Trp into l-Trp-AMP by the adenylation domain of TioK followed by installation of the activated amino acid onto the thiolation domain of this didomain enzyme. However, testing this series of events has been hindered by the inability to heterologously express soluble TioK. Here, we demonstrated that the MbtH-like protein TioT is required for production and activation of TioK. With soluble functional TioK in hand, we established the amino acid substrate profile and kinetically characterized this enzyme. By site-directed mutagenesis of TioT, we also investigated the significance of three Pro residues that are universally conserved in MbtH-like proteins.