Assembly of chimeric glycosyltransferases for directing biosynthesis of natural products

This project seeks to combine the efforts of two established research teams in Cambridge to solve a major outstanding problem in chemistry and biochemistry: how to use protein engineering to generate novel glycosylated compounds that may have important biological activities. A significant fraction of drugs currently used in the clinic, especially anti-infectives such as erythromycins or vancomycins, are natural products isolated from soil bacteria, or derivatives of them. The unusual sugars they contain have been shown to be vital for biological activity. Enzymic modification of natural products is an attractive option to generate new compounds and decorating compounds with different sugars is particularly appealing. One major class of glycosyltransferases (GTs), the enzymes responsible for transferring a sugar from the donor nucleotide diphosphate (NDP) sugar to the acceptor molecule, is known to be composed of two domains. One binds the NDP-sugar donor and the other the acceptor molecule. Our preliminary studies have shown that by cutting and pasting domains from different GTs, hybrid enzymes can be constructed that remain highly active and have the respective donor and acceptor specificity of the parent GTs from which they were derived. This finding has led to the synthesis of several novel vancomycin analogues. These proof of principle experiments pave the way for making a wide range of hybrid GTs from over 19,000 known parent proteins to provide a toolbox of catalysts for accessing novel compounds. Constructing and exploiting these hybrid enzymes will require diverse experimental approaches from carrying out the synthesis of the novel compounds in a test tube with the purified enzymes to genetic manipulation of the genes inside the bacterial cells targetting novel natural products. Success in this project would deliver broad potential benefits to the pharmaceutical industry.

Altering the glycosylation pattern of natural products is potentially one of the most promising ways of accessing novel compounds using combinatorial biochemistry. A limiting factor has been the intrinsic specificity of glycosyltransferases (GTs) for their substrates. To address this we propose to construct hybrid glycosyltransferases to enable custom glycosylation of target compounds. Recent structural studies have shown that all characterised GTs fall into two families. The GT-A family is a single domain protein whereas GT-B proteins are composed of two domains, both with a Rossmann-type chain fold. In all members of the GT-B family studied so far studied the N-terminal domain contains the aglycone acceptor site and the C-terminal domain contains the NDP-sugar donor site. Given the di-domain structure of the GT-B family an attractive strategy for glycodiversification would be to mix and match domains. This would have the advantage that both the sugar and the aglycone could be varied. Using this strategy we have constructed three chimeric GTs from the biosynthetic pathway to the vancomycin family of antibiotics and they showed the expected activity based on the parent enzymes. This has led to the successful synthesis of a vancomycin analogue that would be difficult to obtain using existing glycodiversification techniques. This acts as a proof of principle and suggests this could be a very powerful strategy for glycodiversification. In this proposal we seek to extend this strategy to a wider range of GTs with the aim of understanding the 'rules' for making active hybrids, and creating a toolbox of chimeric GTs for the synthesis of natural products with novel glycosylation profiles.