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bioRxiv · 10.64898/2026.05.26.727834

Systematic prediction and functional analysis of amino acid residues determining product specificity in the plant oxidosqualene cyclase superfamily

Kumari, R.·Sen, N.·Casson, R.·Owen, C.·Stephenson, M.·Borkakoti, N.·Orengo, C.·Thornton, J.·Osbourn, A.
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Abstract

Oxidosqualene cyclases (OSCs) catalyse one of natures most intricate enzyme reactions, converting the linear precursor 2,3-oxidosqualene into an array of cyclic triterpene scaffolds through sequential carbocation cascades. Predicting OSC function based on sequence is challenging beyond broad family-level classification. Here, we develop a structure-based computational framework to identify amino acid determinants of OSC product specificity. Using 169 functionally characterised OSCs, we deploy a multifaceted approach combining differential conservation along with structural information, physico-chemical properties of amino acids and binding pocket electrostatics in order to understand the determinants of product specificity. Using Arabidopsis thaliana cycloartenol synthase AtCAS as a model, we then validate our predictions through targeted mutagenesis, achieving stepwise reprogramming towards the protosteryl-type products cucurbitadienol and lanosterol, including complete product switches. Molecular dynamics simulations support a mechanism in which subtle pocket remodelling alters active-site volume, water access and proton-elimination chemistry. These findings provide a blueprint for OSC engineering.

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Kumari, R., Sen, N., Casson, R., Owen, C., Stephenson, M., Borkakoti, N., Orengo, C., Thornton, J., Osbourn, A.. 2026-05-27. Systematic prediction and functional analysis of amino acid residues determining product specificity in the plant oxidosqualene cyclase superfamily. https://doi.org/10.64898/2026.05.26.727834

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