Reciprocal mutations of two multifunctional β-amyrin synthases fromBarbarea vulgarisshift α/β-amyrin ratios

Abstract

In the wild cruciferous wintercress (Barbarea vulgaris), β-amyrin-derived saponins are involved in resistance against insect herbivores like the major agricultural pest diamondback moth (Plutella xylostella). Enzymes belonging to the 2,3-oxidosqualene cyclase family have been identified and characterized in B. vulgaris G-type and P-type plants that differ in their natural habitat, insect resistance and saponin content. Both G-type and P-type plants possess highly similar 2,3-oxidosqualene cyclase enzymes that mainly produce β-amyrin (Barbarea vulgaris Lupeol synthase 5 G-Type; BvLUP5-G) or α-amyrin (Barbarea vulgaris Lupeol synthase 5 P-Type; BvLUP5-P), respectively. Despite the difference in product formation, the two BvLUP5 enzymes are 98% identical at the amino acid level. This provides a unique opportunity to investigate determinants of product formation, using the B. vulgaris 2,3-oxidosqualene cyclase enzymes as a model for studying amino acid residues that determine differences in product formation. In this study, we identified two amino acid residues at position 121 and 735 that are responsible for the dominant changes in generated product ratios of β-amyrin and α-amyrin in both BvLUP5 enzymes. These amino acid residues have not previously been highlighted as directly involved in 2,3-oxidosqualene cyclase product specificity. Our results highlight the functional diversity and promiscuity of 2,3-oxidosqualene cyclase enzymes. These enzymes serve as important mediators of metabolic plasticity throughout plant evolution.

Genome-Wide Investigation of Oxidosqualene Cyclase Genes Deciphers the Genetic Basis of Triterpene Biosynthesis in Tea Plants

“…Given the substantial evolutionary relatedness of OSC enzymes across plant lineages, a high degree of sequence conservation among these enzymes would be indicative of functional similarities. 36,37 Functional Characterization of CsOSCs. For the functional analysis of all CsOSCs, heterologous expression systems in S. cerevisiae and N. benthamiana were employed.…”

“…sinensis. Given the substantial evolutionary relatedness of OSC enzymes across plant lineages, a high degree of sequence conservation among these enzymes would be indicative of functional similarities. , …”

Adjusting Catalytic Activity of β-Amyrin Synthase GgBAS by Utilizing the Plasticity Residues of an Active Site

“…β-Amyrin synthase (bAS) is a representative plant OSC with a typical CCC fold (chair–chair–chair conformation of the first three fused rings). It is found in various species and is responsible for catalyzing the formation of the prevalent 6/6/6/6/6 pentacyclic scaffold known as β-amyrin (Figure A). − β-Amyrin serves as the precursor for multiple pentacyclic triterpenoids in plants, including oleanolic acid, glycyrrhetnic acid, medicagenic acid, and saponins (Figure B). , Previous investigations into the catalytic mechanisms of bAS have revealed a highly conserved MWCYCR motif at the catalytic center of most plant bASs . Notably, a W259L mutation in the MWCYCR motif of Panax ginsengβ-amyrin synthase has been shown to reprogram the enzyme, resulting in lupeol becoming the major product (lupeol:β-amyrin = 2:1).…”

Insight into neofunctionalization of 2,3‐oxidosqualene cyclases in B,C‐ring‐opened triterpene biosynthesis in quinoa

“…2,3-Oxidosqualene cyclase from plants has been extensively studied and functionally characterized (Thimmappa et al, 2014;Wang et al, 2022). Typical plant OSCs capable of generating monocyclic, fused-ring bi-, tri-, tetra-and pentacyclic triterpenes include the monocyclic camelliol C synthase from Arabidopsis thaliana (Kolesnikova et al, 2007), bicyclic poaceatapetol synthase from rice (Xue et al, 2018b), tricyclic thalianol and arabidiol and tetracyclic tirucalladenol synthase from A. thaliana (Fazio et al, 2004;Xiang et al, 2006;Morlacchi et al, 2009), tetracyclic dammarenediol synthase from Panax ginseng (Tansakul et al, 2006), and pentacyclic b-amyrin synthase (bAS) and cycloartenol synthase (CAS) from numerous species (Salmon et al, 2016;G€ unther et al, 2022;Fig. 1).…”