The sugar donor specificity of plant family 1 glycosyltransferases

Abstract

Plant family 1 glycosyltransferases (UGTs) represent a formidable tool to produce valuable natural and novel glycosides. Their regio- and stereo-specific one-step glycosylation mechanism along with their inherent wide acceptor scope are desirable traits in biotechnology. However, their donor scope and specificity are not well understood. Since different sugars have different properties in vivo and in vitro, the ability to easily glycodiversify target acceptors is desired, and this depends on our improved understanding of the donor binding site. In the aim to unlock the full potential of UGTs, studies have attempted to elucidate the structure-function relationship governing their donor specificity. These efforts have revealed a complex phenomenon, and general principles valid for multiple enzymes are elusive. Here, we review the studies of UGT donor specificity, and attempt to group the information into key concepts which can help shape future research. We zoom in on the family-defining PSPG motif, on two loop residues reported to interact with the C6 position of the sugar, and on the role of active site arginines in donor specificity. We continue to discuss attempts to alter and expand the donor specificity by enzyme engineering, and finally discuss future research directions.

Keywords: donor specificity; enzyme engineering; glycodiversification; glycosyltransferase; structure-function.

SCHEME 1

The general reaction mechanism of UGTs with the catalytic residues presented in blue, the donor substrate in black, and the acceptor substrate (salicylic acid) in green. Here, O-glycosylation is used as an example which includes a deprotonation step.

FIGURE 1

Chemical structures of UDP-sugars which have been discovered to be donor substrates for UGTs. The differences between the UDP-sugars have been marked with a grey circle using UDP-Glc as the reference structure. UDP-apiose was compared to the Haworth projection of UDP-α-d-glucofuranose.

FIGURE 2

(A) A sequence logo generated from a multiple sequence alignment of 224 UGTs, each with determined donor specificity. (B) A view of the donor substrate binding site with the conserved residues of the PSPG motif displayed as grey sticks and UDP-Glc displayed in tan (PDB: 6SU6). The dotted lines represent potential intermolecular interactions between the UGT and UDP-Glc.

FIGURE 3

The residues which have been reported to be important for UDP-sugar donor specificity, displayed in PDB: 6SU6. (A) An overview of the residues displayed as dot spheres representing the van der Waals surface area. Each color corresponds to a specific area: blue indicates the N5 loop, magenta indicates the PSPG motif, and yellow indicates residues near the cavity entrance. (B) A close-up view of the N5 loop area. (C) A close-up view of the PSPG motif area. (D) A close-up view of the residues close to the cavity entrance. “P” refers to the position in the corresponding UGT or PSPG motif mentioned in parentheses.

FIGURE 4

A view of the three different Arg positions described in the text. Alphafold (Jumper et al., 2021) models of UGT94B1, UGT88D4, and VvGT5 were used and had UDP-Glc from PDB 6SU6, displayed in tan, superimposed into the models.