Complete set of glycosyltransferase structures in the calicheamicin biosynthetic pathway reveals the origin of regiospecificity

Abstract

Glycosyltransferases are useful synthetic catalysts for generating natural products with sugar moieties. Although several natural product glycosyltransferase structures have been reported, design principles of glycosyltransferase engineering for the generation of glycodiversified natural products has fallen short of its promise, partly due to a lack of understanding of the relationship between structure and function. Here, we report structures of all four calicheamicin glycosyltransferases (CalG1, CalG2, CalG3, and CalG4), whose catalytic functions are clearly regiospecific. Comparison of these four structures reveals a conserved sugar donor binding motif and the principles of acceptor binding region reshaping. Among them, CalG2 possesses a unique catalytic motif for glycosylation of hydroxylamine. Multiple glycosyltransferase structures in a single natural product biosynthetic pathway are a valuable resource for understanding regiospecific reactions and substrate selectivities and will help future glycosyltransferase engineering.

Structural Studies of the Spinosyn Rhamnosyltransferase, SpnG

“…: 2.6 Å, identity: 28%, PDB code: 3OTI), OleI (Z-score: 34.4, r.m.s.d. : 2.9 Å, identity: 20%, PDB code: 2IYA) (27, 29–31). …”

“…The equivalent loops within the UrdGT2 dimer also exist in two different conformations, and the equivalent loops in the CalG3 dimer (comprised of four glycine residues) are known to reorder upon substrate binding (Figure 4C) (27–29). In the stabilized conformation the S295 side chain forms a hydrogen bond with the backbone carbonyl of W142, which may influence the rotomeric conformations of F315 and D316.…”

“…Structures of several GTs bound to their acceptor substrates are known: OleI+UDP+oleandomycin (PDB code: 2IYA), OleD+UDP+erythromycin (PDB code: 2IYF), GtfD+TDP+desvancosaminyl vancomycin (PDB code: 1RRV), GtfA+TDP+vancomycin (PDB code: 1PN3), vvGT1+UDP-2F-Glucose+UDP-2F (PDB code: 2C1Z), CalG1+calicheamicin+TDP (PDB code: 3OTH), CalG2+TDP+calicheamicin (PDB code: 3RSC), CalG3+calicheamicin+TDP (PDB code: 3OTI) (29–30, 33–34, 36, Glyco3D). The residues between the third and fourth β-strands of the N-terminal domain comprise a “specificity loop” (30).…”

Heteroatom–Heteroatom Bond Formation in Natural Product Biosynthesis

“…Once the N -hydroxyl group is installed, CLM glycosyltransferase CalG2 transfers 27 to the thiosugar, forming an N–O linked disaccharide. 117,118 …”

Understanding substrate selectivity of human UDP-glucuronosyltransferases through QSAR modeling and analysis of homologous enzymes

“…Among the ten bacterial structures, TDP- epi -vancosaminyltransferase GtfA, UDP-glucosyltransferase GtfB and TDP-vancosaminyltransferase GtfD are involved in vancomycin synthesis (Mulichak, et al, 2001, 2003, 2004); four calicheamincin glycosyltransferases (CalG1, CalG2, CalG3, and CalG4) are involved in calicheamicin synthesis (Zhang, et al, 2008; Chang et al, 2011); the oleandomycin glycosyltransferases OleD and OleI inactivate oleandomycin and diverse macrolide antibiotics (Bolam, et al, 2007); and glycosyltransferase UrdGT2 catalyses the formation of a C-C bond between a polyketide aglycone and D-olivose (Mittler, et al, 2007). Although the sequence identity between human UGTs and those GT1 enzymes whose structures have been solved is typically below 20%, the structural fold is predicted to be highly conserved (Coutinho et al, 2003; Breton et al, 2006).…”