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

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Abstract
Oxidosqualene cyclases (OSCs) catalyse one of nature's 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.
Funding Information
  • BBSRC (BB/V015176/1)
  • BBSRC (BB/S020144/1)
  • BBSRC (BB/S016023/1)
  • BBSRC (BB/X01097X/1)
  • Novo Nordisk Foundation (Novozymes Prize 2023)
  • Wellcome Trust (#227375/Z/23/Z)
  • John Innes Foundation