Periodic Reporting for period 2 - CARBOCENTRE (Activity-Based Profiling of Glycoprocessing Enzymes for Human Health and a Sustainable Society)
Reporting period: 2022-12-01 to 2024-05-31
Oligosaccharides and glycoconjugates constitute the most abundant and diverse class of biomolecules on Earth. The enzymes that produce and degrade these glycan structures are present in all kingdoms of life. The ability to visualize, modulate and understand these carbohydrate-active enzymes (CAZymes) therefore offers great potential for human health and sustainable industries. However, this potential is under-realised because of a lack of CAZyme-specific molecular tools, which can be used to discover and to perturb glycoprocessing enzymes both in vivo and in complex biotechnological milieu. To provide a “disruptive” shift in our understanding, Carbocentre follows a multidisciplinary approach combining structural biology, enzymology, computational chemistry, organic synthesis, and chemical biology. Three fundamental strands will specifically target and ‘capture’ the active sites of the three main glycoprocessing enzyme families: retaining glycosidases (strand one), inverting glycosidases (strand 2) and glycosyltransferases (strand 3). Biochemical and 3-D structural analyses will inform computational dissection of the reaction coordinate of key enzymes for human health and biotechnology processes. This knowledgebase will be used to inform the rational design and synthesis of probes and inhibitors, utilising fluorescent, bio-orthogonal and capture tags to image, manipulate and discover enzymes. Our probes feeds research in two major application domains of human health and biotechnology – areas that at a first glance appear unrelated but in which glycoprocessing enzymes are heavily involved:
1. To provide visualization, diagnosis, and inhibitor assays and clinical lead compounds for enzymes in cancers and genetic diseases (lysosomal storage disorders), and
2. To explore the natural diversity of CAZymes and to discover, quantify and optimize new enzymes for food and household applications and for biomass conversion to biofuels.
We routinely share our reagents and tools with the international glycobiology community, with on average one to two shipments per month of specific reagents (both those developed within the Carbocentre program and those developed previously) going out to academic groups world-wide. In this way – besides adhering to the open science policy – we increase the impact of the general work ethos that is behind the Carbocentre research, which is to move from 3-D structure through reaction mechanism dissection and compound conformational analysis/prediction and to apply this to probe design, synthesis and application
1. To provide visualization, diagnosis, and inhibitor assays and clinical lead compounds for enzymes in cancers and genetic diseases (lysosomal storage disorders), and
2. To explore the natural diversity of CAZymes and to discover, quantify and optimize new enzymes for food and household applications and for biomass conversion to biofuels.
We routinely share our reagents and tools with the international glycobiology community, with on average one to two shipments per month of specific reagents (both those developed within the Carbocentre program and those developed previously) going out to academic groups world-wide. In this way – besides adhering to the open science policy – we increase the impact of the general work ethos that is behind the Carbocentre research, which is to move from 3-D structure through reaction mechanism dissection and compound conformational analysis/prediction and to apply this to probe design, synthesis and application
The program is divided into fundamental research strands on retaining glycosidases, on inverting ones and on glycosyltransferases. Reagents and probes emerging from these strands are then applied in the areas of biotechnology, with the aim to discover new enzyme activities for the sustainable turnover of biomass polysaccharides for sustainable energy and chemistry. They are also applied in biomedicine, where bespoke reagents may find application in the diagnosis of diseases where carbohydrate-processing enzymes are causative, as well as in identifying (as read-outs in assays, or by functioning a starting-points for drug discovery themselves) enzyme inhibitors/stabilizers for drug development. The three strands tackle enzyme families of increasing complexity, considering the design of active site inactivators: from retaining glycosidases (‘easy’; strand one) over inverting ones (no design blueprint available at the onset of the program; strand two) to glycosyltransferases (no general inhibitor design known at all, be it competitive or covalent; strand three). This is also reflected in translating reagents from the three strands to the domains of application. Most progress is made in strand one on retaining glycosidases, with the first influenza neuraminidase probes realized and design blueprints for the other glycosidases targeted established. As to research stand two on inverting glycosidases, we have the first putative scaffolds calculated and are in the process of preparing these, whereas the first libraries of putative GT inhibitors (competitive ones in the first instance) have been prepared (and await enzyme activity assessment) based on our retaining glycosidase Michaelis conformer mimetics. Work in the biomedicine arena includes enzyme activity assays for diagnosis (measurement of active neuraminidases in influenza vaccines) and drug discovery (we have selective retaining glucosidase inhibitors and are working on inverting ones, and the two combined would allow discriminating between ER alpha-glucosidase I and II in the context of anti-HIV and/or anti-Covid therapeutics development – manuscript in preparation). Work in the biotechnology area includes the successful establishment of workflows that allow detection of various endo- and exoglycosidases, conditionally expressed and excreted by Cellvibrio japonicus depending on the biomass polysaccharide source, in a multiplexing assay format (https://doi.org/10.26434/chemrxiv-2023-lxghm(opens in new window)).
Our collaborator Sarel Fleishman (Weizzman Institute of Technology Israel) has developed a self-learning algorithm with which protein structure databases are mined for secondary structure elements from which de novo glycoprocessing enzyme libraries are generated. These in silico collections (which may run up to 1 million) are then cloned and expressed in yeast, and active enzymes are discriminated from non-active ones using our glycoprocessing enzyme probes. Proof-of-concept is achieved in the design of new xylanase activities (DOI: 10.1126/science.ade9434) and it is our expectation that our tools may assist in the likewise design of new enzyme families, in particular also glycosidases that form new products (‘unnatural’ oligosaccharides) from the same biomass polysaccharide feedstocks, ‘simply’ by imbuing the desired product in the corresponding glycoprocessing enzyme-directed activity-based probe. This development, in which our probes support research by others that is not intrinsically, and conceptually, part of the Carbocentre program, shows the value and unbiased nature of our reagents, which report on glycoprocessing enzyme activity irrespective of their origin or purpose. Another area in which our probes find ready application is in profiling of human gut microbiota on the presence of glycosidases. As an example, our retaining beta-glucuronidase probes have been put to use by Matthew Redinbo (University of North Carolina, USA) in the identification of patients running at risk of regenerating, through the action of gut microbiota-specific retaining beta-glucuronidases, toxic drugs in their intestines (https://doi.org/10.1038/s41467-021-27762-y(opens in new window) and DOI: 10.1080/19490976.2022.2107289).