Functional and structural characterisation of unusual carbohydrate-processing enzymes in natural product biosynthesis
Lead Research Organisation:
University of Warwick
Department Name: School of Life Sciences
Abstract
Many bioactive natural products currently utilised as anticancer and antibiotic agents are glycosylated. The presence of carbohydrate moieties in these molecules is essential to their medicinal activity. Microorganisms that produce natural products harbor innumerous intriguing carbohydrate- processing enzymes, including glycosyltransferases, which are responsible for the making of unusual sugars and their ultimate attachment to natural product aglycones of various origins (e.g. polyketides and nonribosomal peptides).
The detailed elucidation of these enzymes catalytic mechanisms and structural architecture constitutes the basis for the development of novel biotacalysts and of novel synthetic biology approaches towards the generation of 'unnatural' products- molecules that are still produced biosynthetically but that are structurally diversified (e.g. differently glycosylated) from their original counterparts, and may be of superior value.
The aim of this PhD project is to elucidate the function and the structure of biosynthetic enzymes involved in unusual natural product glycosylation. The outcomes of this work will constitute the basis for the development of novel biocatalytic routes to diversified natural products.
The detailed elucidation of these enzymes catalytic mechanisms and structural architecture constitutes the basis for the development of novel biotacalysts and of novel synthetic biology approaches towards the generation of 'unnatural' products- molecules that are still produced biosynthetically but that are structurally diversified (e.g. differently glycosylated) from their original counterparts, and may be of superior value.
The aim of this PhD project is to elucidate the function and the structure of biosynthetic enzymes involved in unusual natural product glycosylation. The outcomes of this work will constitute the basis for the development of novel biocatalytic routes to diversified natural products.
Organisations
| Description | The main outcome so far has been the structural modelling of a putative hexose dehydratase (believed to effectively remove a water from a sugar molecule) from Rhodococcus jostii PR4. This was done using protein crystallography, which allows us to determine where the atoms of the protein sit in relation to each other. Working is on going to characterise the activity of this protein, looking at which different sugar structures it is able to remove water from. By studying the protein structure in this detail and comparing it to other similar characterised proteins, we can determine which features are retained which will help guide experiments and determine the catalytic function of the protein. This grant has also funded work into the structural and functional characterisation of a glycosyltransferase from Actinomadura spp. (currently on going and no significant results to report just yet) and the genomic sequencing of Streptomyces hygroscopigus (a known antibiotic producing strain that has yet to be genomically sequenced, the sequencing will allow genes/proteins responsible for the production of the antibiotic structure to be analysed and characterised). Other proteins were initially pursued as part of this grant (e.g. a glycosyltransferase and sugar modifying enzymes from Streptomyces cattleya, glycosytransferases from Streptomyces nanchangensis and Streptomyces carzinostaticus) but work as stopped as these proteins were determined to be insoluble, making them extremely difficult to characterise. |
| Exploitation Route | Characterising these enzymes will allow them to be used in biosynthetic pathways to produce natural product derivatives or 'unnatural' products. These types of product often possess significant pharmacological activity, such as antibiotic, antiviral, antifungal or antitumour. We are focussing on glycosyltransferases and sugar modifiers/carbohydrate processing enzymes as the incorporation of sugar moieties into these natural products can influence their solubility, cellular uptake and molecular recognition and improve their potency. Once the enzymes are characterised, they can be used in either their wild type form (where they can be used with non-native substrates or the enzymatic products can be combined with alternative chemical scaffolds to produce novel glycosylated natural products) or a mutated form (changing specific amino acids within the protein to manipulate the substrate tolerance or catalytic ability of the enzyme to produce novel or uncommon sugar structures). |
| Sectors | Pharmaceuticals and Medical Biotechnology |
| Title | Protein model of RER_RS03965 from Rhodococcus jostii PR4 |
| Description | Structural model of a putative nucleotide diphosphate hexose dehydratase (NDPHD) protein from Rhodococcus jostii PR4. Gene/orf associated with this protein denoted RER_RS03965. Model has a resolution of 1.7 angstrom and an Rfree value of 0.22 (indicator of model quality/accuracy, lower value indicated better quality). Data currently unpublished, awaiting functional characterisation of protein (in progress). |
| Type Of Material | Computer model/algorithm |
| Year Produced | 2021 |
| Provided To Others? | No |
| Impact | Model will help with elucidating the substrate preference/specificity and catalytic ability of the protein. |