Structural characterization of O- and C-glycosylating variants of the landomycin glycosyltransferase LanGT2

Abstract

The structures of the O-glycosyltransferase LanGT2 and the engineered, C-C bond-forming variant LanGT2S8Ac show how the replacement of a single loop can change the functionality of the enzyme. Crystal structures of the enzymes in complex with a nonhydrolyzable nucleotide-sugar analogue revealed that there is a conformational transition to create the binding sites for the aglycon substrate. This induced-fit transition was explored by molecular docking experiments with various aglycon substrates.

Keywords: C-glycosylation; Friedel-Crafts alkylation; carbasugars; enzyme engineering; glycosyltransferases.

Figure 1

Structures of the angucycline antibiotics landomycin A and urdamycin A. The O-GT LanGT2 attaches D-olivose to the aglycon through an O-glycosidic bond, while the C-GT UrdGT2 links the same carbohydrate moiety through a C—C bond to the aglycon of urdamycin A.

Figure 2

Three-dimensional structures of LanGT2. A) The LanGT2 monomer colored from blue at the N terminus to red at the C terminus. The protein is organized as a nucleotide-sugar-binding domain (top) and an aglycon-binding domain (bottom) that are connected through a flexible hinge. B) A 90° rotation highlights the grafted loop at the rim of the N-terminal domain. C) Detail of the grafted region in LanGT2 (red), LanGT2S8Ac (blue), and UrdGT2 (purple, PDB-ID 2P6P). The UrdGT2-derived region retains its conformation in the LanGTS8Ac chimera. The box in (B) highlights this region in LanGT2.

Figure 3

Ligand binding to LanGT2. A) Stereo image of a C_α-trace superposition of unbound LanGT2 (black) and the enzyme with the synthetic analogue TDP-carba-D-olivose (TcO, red) bound. The ligand induces a 10° rotation of the sugar-binding domain relative to the aglycon-binding domain (grey arrow). B) Stereo representation of the active site of LanGT2 with bound TcO. While the loop region 267–271 assures specific binding of the TDP moiety, the negatively charged phosphodiester is stabilized by the helix dipole of helix h10 (residues 286–295). The 4′ and 5′ hydroxy groups of olivose are recognized by G286 and D137, with the latter being the only residue from the N-terminal domain that is involved in binding.

Figure 4

In silico analysis of aglycon binding. A) Stereo image of a docking solution for deoxylandomycinone and LanGT2. In the model, the ligand (shown as van der Waals spheres) displaces R220 and R59 and occupies the broad cleft formed by loop region 51–62 (white). B) In LanGT2S8Ac, the 51–62 loop was replaced by the UrdGT2 sequence (white), thus effectively closing the substrate-binding cleft. Molecular docking suggests that the ligand tetrangulol attains a different binding mode that reorients R220 towards the substrate and TDP-carbaolivose (TcO), thus resulting in a major repositioning of the ligand. In the orientation chosen, the catalytic base D137 is located behind the ligand.

Scheme 1

Synthesis of α-D-carbaolivose thymidine diphosphate (8). acac =acetylacetonate, BOP =benzotriazol-1-yloxytris(dimethylamino)phosphonium, O-isoval =isovalerate, LAH =lithium aluminum hydride, THF =tetrahydrofuran, TMS =trimethysilyl.