Crystal Structures of the Glycopeptide Sulfotransferase Teg 12 Complexed with the Teicoplanin Aglycone

The TEG gene cluster, a glycopeptide biosynthetic gene cluster that is predicted to encode the biosynthesis of a polysulfated glycopeptide congener, was recently cloned from DNA extracted directly from desert soil. This predicted glycopeptide gene cluster contains three closely related sulfotransferases (Teg12, 13, and 14) that sulfate teicoplanin-like glycopeptides at three unique sites. Here we report a series of structures including: an apo structure of Teg12, Teg12 bound to the desulfated co-substrate 3'-phosphoadenosine 5'-phosphate and Teg12 bound to the teicoplanin aglycone. Teg12 appears to undergo a series of significant conformational rearrangements during glycopeptide recruitment, binding and catalysis. Loop regions that exhibit the most conformational flexibility show the least sequence conservation between TEG sulfotransferases. Site directed mutagenesis guided by our structural studies confirmed the importance of key catalytic residues as well as the importance of residues found throughout the conformationally flexible loop regions. During the 20th century, widespread use of antibiotics significantly reduced the threat of many once lethal infectious diseases. However, the success of these wonder drugs may soon become their Achilles’ heel. Bacterial pathogens that have developed resistance to most widely used antibiotics are now regularly seen in clinical settings. Vancomycin and teicoplanin are glycopeptide antibiotics used in the treatment of many gram-positive bacterial infections, including methicillin-resistant Staphylococcus aureus (MRSA). With the appearance of vancomycin resistant Enterococci in the late 1980s and resistant Staphylococci in the early 1990s, these traditional antibiotics of last resort are in danger of becoming clinically compromised (1–3). As with many bacterial natural products, the discovery of additional glycopeptide congeners that might combat the growing problem of antibiotic resistance has slowed as it has become increasingly difficult to identify new biodiversity from which novel molecules might be characterized. The vast majority of bacteria present in the environment remain recalcitrant to culturing (4). This uncultured majority no doubt contains previously inaccessible glycopeptide biosynthetic gene clusters, many of which could encode the biosynthesis of novel glycopeptide congeners. Although metabolites produced by bacteria that are difficult to culture in the laboratory cannot be characterized using standard microbiological methods, it is possible to extract DNA directly from environmental samples and then analyze this DNA for sequences that might encode the biosynthesis of new natural products. In a recent analysis of DNA extracted directly from desert soil, we uncovered a new glycopeptide biosynthetic gene cluster (the TEG gene cluster) that is predicted to encode the biosynthesis of the first polysulfated glycopeptide congeners (5). The TEG gene cluster contains three closely related 3'-phosphoadenosine 5'-phosphosulfate (PAPS) dependent sulfotransferases (Teg12, 13 and 14). In vitro, these three sulfotransferase *To whom correspondence should be addressed. Sean F. Brady: sbrady@rockefeller.edu. Phone: 212-327-7478. Fax: 212-327-8281. NIH Public Access