Crystal Structures of the Glycopeptide Sulfotransferase Teg12 in a Complex with the Teicoplanin Aglycone

Abstract

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 20 th 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 sulfotransfera...

Sulfonation of glycopeptide antibiotics by sulfotransferase StaL depends on conformational flexibility of aglycone scaffold

“…As stated above, four highly similar GPA sulfotransferases (StaL, Teg12, Teg13, and Teg14) regioselectively modify unique residues on the teicoplanin-class GPA scaffold. Sequence alignment indicates that these proteins differ mainly in three variable regions (V1, V2, and V3) (17,18). Our current StaL-PAP-DSA structure confirms that the DSA substrate, bound in a productive mode within the active site, interacts with all of these three regions, loops α2/α3 (V1), α6/α7 (V2), and α12/α13 (V3) (SI Appendix, Fig.…”

“…The sulfotransferase enzymes within the Teg cluster share ∼60% sequence identity with StaL, and all use the 3′-phosphoadenosine 5′-phosphosulfate (PAPS) cofactor as the source of the sulfate group, converting it to the product 3′-phosphoadenosine 5′-phosphate (PAP) after transfer to the GPA substrate. The crystal structures of Teg12 complexes (17) and Teg14 apo protein (18) have been reported recently. Their structural similarity to StaL, expected given their sequence similarity, implies that each of these orthologs binds the GPA aglycone in a different way, exposing different parts of the GPA aglycone to the enzyme active site.…”

Three‐dimensional structure of cyclic antibiotic teicoplanin aglycone using NMR distance and dihedral angle restraints in a DMSO solvation model

“…7). [28] These results are consistent with our findings regarding the flexibility of the G and F portions of teicoplanin aglycone.…”

“…The flexibility of this part of the molecule is evident from an X-ray structure of the aglycone bound to sulfotransferase Teg12. [28] Teg12 is a protein known to sulfate teicoplanin-like glycopeptides and is reported to undergo a series of significant conformational rearrangements during glycopeptide recruitment, binding, and catalysis when complexed with teicoplanin aglycone molecules. Likewise, teicoplanin aglycone shows considerable flexibility at rings G and F upon binding to Teg 12.…”

Structural Basis of Functional Group Activation by Sulfotransferases in Complex Metabolic Pathways

“…Natural products pathways do not include exclusively STs of the activating ST family. For example the glycopeptide ST Teg12 (29) (16% identity to CurM ST) clearly belongs to a different ST family.…”