Structure of the TDP-epi-vancosaminyltransferase GtfA from the chloroeremomycin biosynthetic pathway

Abstract

During the biosynthesis of the vancomycin-class antibiotic chloroeremomycin, TDP-epi-vancosaminyltransferase GtfA catalyzes the attachment of 4-epi-vancosamine from a TDP donor to the beta-OHTyr-6 of the aglycone cosubstrate. Glycosyltransferases from this pathway are potential tools for the combinatorial design of new antibiotics that are effective against vancomycin-resistant bacterial strains. These enzymes are members of the GT-B glycosyltransferase superfamily, which share a homologous bidomain topology. We present the 2.8-A crystal structures of GtfA complexes with vancomycin and the natural monoglycosylated peptide substrate, representing the first direct observation of acceptor substrate binding among closely related glycosyltransferases. The acceptor substrates bind to the N-terminal domain such that the aglycone substrate's reactive hydroxyl group hydrogen bonds to the side chains of Ser-10 and Asp-13, thus identifying these as residues of potential catalytic importance. As well as an open form of the enzyme, the crystal structures have revealed a closed form in which a TDP ligand is bound at a donor substrate site in the interdomain cleft, thereby illustrating not only binding interactions, but the conformational changes in the enzyme that accompany substrate binding.

Fig. 1.

Structure and glycosylation pattern of chloroeremomycin. The Gtfs catalyzing sugar attachments are shown in bold; in parentheses are the corresponding Gtfs from the biosynthesis of vancomycin, which has only the disaccharide attachment at residue 4.

Fig. 2.

GtfA catalyzes the addition of 4-epi-vancosamine to the DVV substrate. (a) HPLC traces show the production of 4-epi-balhimycin only in the presence of bGtfA. The assay condition is described in Materials and Methods. (b) Schematic representation of the conversion of DVV to 4-epi-balhimycin.

Fig. 3.

(a) Stereoview comparing the open (blue) and closed (green) forms of GtfA, with bound TDP (gold) and DVV (red) as observed in closed conformation. The flexible loop, unobserved in open form, is shown in magenta. (b) An expanded view looking into cleft of closed form (molecule B). Positions of bound DVV (red) and vancomycin (blue) are superimposed, highlighting the altered binding at the glycosylation site (arrow). The figure was prepared by using molscript (27) and raster3d (28).

Fig. 4.

(a) Stereoview showing difference electron density (3σ contour level) for DVV bound to molecule A. Highlighted are the putative catalytic residues Ser-10 and Asp-13 (in gold) and the side chains binding glucose moiety (in blue). (b) Molecular surface view of the GtfA ternary complex (molecule B). DVV binds against surface of N-terminal domain with the attacking hydroxyl (arrow) pointed toward the β-phosphate of TDP. The relative positions of the potential catalytic residues (green), hypervariable regions in the N-terminal domain (blue), and poorly ordered loop in the C-terminal domain (yellow) are shown. The image in a was prepared by using setor (29). The image in b was prepared by using spock (30) and raster3d (28).

Fig. 5.

(a) Difference electron density (3σ level) for TDP in the interdomain cleft. (b) Binding interactions of TDP (gold) with residues of the N-terminal (cyan) and C-terminal (green) domains including the ²⁹²HHXXAGT²⁹⁸ loop. Ribose moiety interacts only via buried water molecules (blue spheres). Interdomain aromatic capping interaction between Arg-11 and Glu-277 and position of attacking hydroxyl (asterisk) of the bound DVV (red) are also shown. The images were prepared by using setor (29).