Abstract
Enzyme catalysis has emerged as a key technology for developing efficient, sustainable processes in the chemical, biotechnological and pharmaceutical industries. Plants provide large and diverse pools of biosynthetic enzymes that facilitate complex reactions, such as the formation of intricate terpene carbon skeletons, with exquisite specificity. High-resolution structural analysis of these enzymes is crucial in order to understand their mechanisms and modulate their properties by targeted engineering. Although cryo-electron microscopy (cryoEM) has revolutionized structural biology, its applicability to high-resolution structural analysis of comparatively small enzymes has so far been largely unexplored. Here, it is shown that cryoEM can reveal the structures of plant borneol dehydrogenases of ∼120 kDa at or below 2 Å resolution, paving the way for the rapid development of new biocatalysts that can provide access to bioactive terpenes and terpenoids.
Highlights
Despite the stunning recent success of single-particle cryo-electron microscopy (cryoEM) in the structural analysis of many large molecular machines, comparatively small proteins remain a major challenge for this technique (Kuhlbrandt, 2014; Lyumkis, 2019; Vinothkumar & Henderson, 2016)
Encouraged by the possible occurrence of an octameric assembly, we considered cryoEM as powerful method to dissect structural heterogeneity, and prepared cryoEM grids
Given the molecular mass of the tetrameric complex, here we report the highest resolution achieved by cryoEM so far (Supplementary Table S1), pushing the boundaries of this rapidly developing method
Summary
Despite the stunning recent success of single-particle cryoEM in the structural analysis of many large molecular machines, comparatively small proteins remain a major challenge for this technique (Kuhlbrandt, 2014; Lyumkis, 2019; Vinothkumar & Henderson, 2016). Protein engineering relies mainly on directed evolution or semi-rational approaches While often successful, these methods require the implementation of high-throughput screenings and a substantial effort in terms of laboratory work, leading to long time-to-market horizons. The outstanding selectivity of the enzymes involved in the formation of terpene carbon skeletons (Christianson, 2017), their primary functionalization (Bohlmann & Keeling, 2008) and their further derivatization (Rinkel et al, 2019) could enable the formation of a myriad of new terpene derivatives with diverse, interesting properties for the food and pharmaceutical industries via environmentally friendly catalytic processes (Newman & Cragg, 2016; Oldfield & Lin, 2012) To this end, a detailed understanding of the molecular basis of the reaction mechanisms and selectivity of the enzymes is required. We report the determination of the structures of two stereoselective dehydrogenases, SrBDH1 and SoBDH2, by single-particle cryoEM
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