Towards the structure-informed engineering of enzymes in the avenacin biosynthesis pathway

Pfalzgraf, Hans (2022) Towards the structure-informed engineering of enzymes in the avenacin biosynthesis pathway. Doctoral thesis, University of East Anglia.

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Glycosylated triterpenes represent numerous and diverse plant natural products, but difficult production has limited their applications in health, food and industry.

This thesis describes the structural characterisation of three enzymes from the biosynthetic pathway of avenacin, an antifungal glycosylated triterpene from oat, to enable their rational engineering.

Avena strigosa arabinosyltransferase (AsAAT1) and transglucosidase (AsTG1), involved in the glycosylation of avenacin, were expressed in Escherichia coli and purified, but did not crystallise. Several deletion constructs proved insoluble, so molecular models were used to rationalise both the specificity of three AsAAT1 mutants for various sugar donors and the switch from glucosyl hydrolase to transglucosidase activity in AsTG1, which was suggested by a multiple sequence alignment. Molecular dynamics simulations of AsTG1 confirmed its ability to discriminate between analogous substrates.

The membrane-bound A. strigosa β-amyrin synthase (AsbAS1), which forms the triterpene scaffold of avenacin, was expressed in E. coli. Attempts to purify and crystallise it only led to the high-resolution structure of a contaminant, HPII catalase. AsbAS1 mutants were designed, inspired by a soluble homologue, to simplify this process. With no solubilised protein observed in E. coli, AsbAS1 was expressed in the yeast Pichia pastoris instead, which resulted in active protein. A homologue, Euphorbia tirucalli β-amyrin synthase (EtAS), was expressed in an active form in E. coli. These expression methods could be used to produce two different β-amyrin synthases and attempt to obtain the first crystal structure of a plant oxidosqualene cyclase. A multiple sequence alignment and models of other homologues generated with AlphaFold2 enabled the design of four AsbAS1 mutants that may have altered product specificity.

This work shows structural information for three enzymes in the avenacin biosynthesis pathway, leading to the rationalisation of the effect from various amino acids. This can now be tested by expressing mutants using the methods described.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Chris White
Date Deposited: 22 Mar 2023 15:09
Last Modified: 22 Mar 2023 15:09

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