Mapping C-C nucleoside bond formation in real time

Lead Research Organisation: Cardiff University
Department Name: Chemistry

Abstract

We have set out to study an important new enzyme which makes a bond between two carbon atoms. The making of carbon carbon bonds is both the chemistry of life at its most basic and it is the central pillar of enzymatic chemistry. Being able to use an enzyme to make this bond, will make it possible to make new drugs. We have targeted so called C-nucleosides. These molecules trick bacteria and viruses into using them instead of the natural N-nucleosides. As a result of using these molecules, viruses and bacteria are killed. The carbon carbon bond is absolutely key to these molecules, it prevents the bacteria and virus from degrading the compound. These C-nucleosides are used by nature but they are hard to make, hence the need for new ways such as enzymes to make them. The system we are studying is also appealing because we can use it to study the grand challenge of watching chemistry in real time. Chemist occurs in around 1 billionth of second, far to fast for most techniques to see but the new X-ray free electron laser finishes experiments in around 1 trillionth of second, allowing us to see for the first time an enzyme catalysed chemical reaction. This ground breaking experiment will require a lot of preparation and new technologies which the grant will develop. Being able to see in real time a chemical reaction is a grand challenge of chemistry, biochemistry and biology. It will change how we think and what we know.

Technical Summary

Natural products, once the source of all medicines, fell from favour for a number of reasons, complex chemical structures (eg multiple stereocentres), problematic chemical synthesis, and limited abundance. Optimising their pharmacokinetic properties by chemical modification can be impossible. Yet, natural products have evolved to enter cells, and their structures are recognized as "privileged" scaffolds. Technological advances in genome sequencing and protein engineering offer new tools to take build upon these scaffolds for drug discovery. The heterologous expression of engineered pathways can yield large quantities of product for biological testing and/or chemical modification .We have solved two structures and about to solve a third, the C-nucleoside forming enzyme (ForS) and its homologue RHP synthase (MJ1427), an enzyme from the methanopterin biosynthetic pathway in Methanocaldococcus jannaschii. These enzymes catalyse C-C bond formation, RHP links p-hydroxybenzoic to 5'-phosphoribosyl-1'-pyrophosphate with the concomitant release of CO2 (Fig 2A). Bioinformatic analysis strongly suggests that ForS and RHP synthase are an entirely new class of C-C bond forming enzyme. The substrates used by these enzymes lend themselves to photochemical control, opening up the use of the X-ray Free Electron Laser (XFEL) to map the enzyme-catalysed reaction in real time. This interdisciplinary project, firmly anchored by preliminary data and proven scientific researchers, will deliver new world leading bioscience, new fundamental, enabling technologies for medicinal chemistry, novel C-nucleosides, and a breakthrough in the experimental characterisation of enzyme catalysis. These outcomes will be delivered by pursuing three interdisciplinary work packages.

Planned Impact

The project aims to characterise a new class of carbon-carbon bond-forming enzymes. This project will benefit scientific research, industry, medicinal chemistry and human heath.

Scientific Research
The research will generate new knowledge of the significant central carbon carbon bond forming reaction of formycin. The biosynthesis of formycin is unknown and yet it is a particular well known molecule. Our work will transform our knowledge of carbon carbon bond formation. We will leverage the extraordinary investment in the European XFEL to produce the first "freeze-frame" movie of enzyme catalysis. These studies will not only provide chemical biology tools and information of considerable value to researchers in both industry and academia but also and grow the critical mass of researchers with training in cutting edge time-resolved structural biology. The time resolved work will encourage others by showing new approaches to XFEL structural biology. We will publish this research and deposit our regents to ensure the widest possible uptake.

Industry
The making of carbon carbon bonds is central to biotechnology, it lies behind making bigger molecules and is a serious chemical challenge. Enzymes make C-C bonds in a regioselective and stereoselective manner, making them valuable. We will engineer enzymes capable to make carbon carbon bonds with different substrates than the native enzymes. We will ensure these modified enzymes are available for industrial use.

Medicinal Chemistry
C-nucleosides are a class of molecules exhibiting antiviral, antibacterial and anticancer properties. Our work will create new ways to make these molecules in a library format. This will help medicinal chemists to test new analogues.

Human Health
In the longer term, new C-nucleosides could become new medicines. These molecules are already widely used in nature to kill drug resistant bacteria. It has recently been shown that they are potent antivirals. Our work will create new ways to make libraries of these very important molecules.

Publications

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Description Working in collaboration with Prof. James Naismith and co-workers, we were able to obtain the first high-resolution X-ray crystal structure of ForT complexed to PRPP (Gao et al., Chemical Communications, 2020). The enzyme ForT catalyses a key step in the biosynthesis of formycin. These results provide a firm foundation for reengineering the substrate specificity of this enzyme (Aim 2) and for carrying out time-resolved studies of catalytic mechanism (Aim 3).
Exploitation Route Our findings provide an opportunity to develop biocatalysts that can generate novel nucleosides with potential anti-viral, antibiotic and anti-cancer activities.
Sectors Education,Pharmaceuticals and Medical Biotechnology