Identification of the substrate binding sites in Actinobacteria sulfotransferase Cpz8

Full text: The understanding of natural product biosynthetic pathways has received increased interest of the modern scientific community. Recently, our collaborators at the University of Tubingen (Germany) identified steps in the biosynthesis of the antibiotic caprazamicin (CPZ) in Streptomyces sp.. The CPZ sulfation pathway is catalyzed by a PAPS dependent sulfotransferase, Cpz8. Cpz8 uses PAPS as sulfate donor and a presuficidins as sulfate acceptors. Interestingly, Cpz8 does not display a typical sequence for PAPS binding domain. Low similarity is displayed by Cpz8 when compared to known sulfotransferases (19 and 29% similarity for human and S. mansoni respectively). Therefore, we attempted to determine the crystal structure of Cpz8 to elucidate its mechanism of catalysis and substrate binding. For this, the enzyme was heterologously expressed, purified and subjected to crystallization at RoboLab (LNBio-CNPEM). Diffraction data sets were collected at the MX-2 beam line (LNLS-CNPEM) with resolution better than 2 Å. It was not possible to determine the crystal structure of Cpz8 by molecular replacement. Therefore, SAD experiments were carried out also at the MX-2, using quick cryo-soaking with Nal, CsCl and GdCl₃. After data processing, the phases were recovered using SAD data sets with iodine. The calculated electron density map allowed for the determination of the crystal structure of Cpz8. The atomic model was refined and analyzed. It was found that - despite the poor similarity with known PAPS binding motives in the primary sequence - the Cpz8 3D-structure presents a large cavity compatible with the PAPS binding site. The important chemical groups to interact with PAPS and sulfate transfer are preserved in this putative binding site. A second cavity is also found and predicted to bind the sulfate acceptors. The crystallization of Cpz8 in complex with PAPS and the sulfate acceptor (presuficidin A-D) are in progress. (author)