A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases

Salmon, Melissa; Thimmappa, Ramesha; Minto, Robert E.; Melton, Rachel E.; Hughes, Richard K.; O’Maille, Paul E.; Hemmings, Andrew M.; Osbourn, Anne

doi:10.1073/pnas.1605509113

Cited by 55 publications

(66 citation statements)

References 57 publications

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“…These enzymes produce tetracyclic and pentacyclic triterpenes from the same protosteryl‐type C‐B‐C conformation (Banta et al ., ). Studies (Ito et al ., ; Salmon et al ., ) on Euphorbia tirucalli and Avena strigosa β ‐ amyrin synthase (SAD1) have identified two conserved residues at positions F696 and S699 (positions of HsLAS) which, when mutated, result in a change in product from the pentacyclic triterpenoid β ‐ amyrin to tetracyclic cyclization products, but maintain a dammarane‐type C‐C‐C conformation. The three key residue positions, #124, #365 and #732, in OsPS and OsOS, corresponding to L104, I335 and I702 in HsLAS, that have been identified here were not reported in the previous studies (Ito et al ., ; Banta et al ., ; Salmon et al ., ).…”

Section: Discussionmentioning

confidence: 99%

Identification of key amino acid residues determining product specificity of 2,3‐oxidosqualene cyclase in Oryza species

et al. 2018

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Triterpene synthases, also known as 2,3-oxidosqualene cyclases (OSCs), synthesize diverse triterpene skeletons that form the basis of an array of functionally divergent steroids and triterpenoids. Tetracyclic and pentacyclic triterpene skeletons are synthesized via protosteryl and dammarenyl cations, respectively. The mechanism of conversion between two scaffolds is not well understood. Here, we report a promiscuous OSC from rice (Oryza sativa) (OsOS) that synthesizes a novel pentacyclic triterpene orysatinol as its main product. The OsOS gene is widely distributed in indica subspecies of cultivated rice and in wild rice accessions. Previously, we have characterized a different OSC, OsPS, a tetracyclic parkeol synthase found in japonica subspecies. Phylogenetic and protein structural analyses identified three key amino acid residues (#732, #365, #124) amongst 46 polymorphic sites that determine functional conversion between OsPS and OsOS, specifically, the chair-semi(chair)-chair and chair-boat-chair interconversions. The different orientation of a fourth amino acid residue Y257 was shown to be important for functional conversion The discovery of orysatinol unlocks a new path to triterpene diversity in nature. Our findings also reveal mechanistic insights into the cyclization of oxidosqualene into tetra- and pentacyclic skeletons, and provide a new strategy to identify key residues determining OSC specificity.

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Section: Discussionmentioning

confidence: 99%

Identification of key amino acid residues determining product specificity of 2,3‐oxidosqualene cyclase in Oryza species

et al. 2018

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“…However, the peak corresponding to tetracyclic triterpenols was of greater area (Figs b, S5). The peak at 23.9 min was identified as cycloartenol by comparison with the authentic cycloartenol standard, and one minor peak at 28.0 min was identified as a putative damarenediol‐II by comparison of its mass fragmentation pattern with a prior report (Salmon et al ., ). These results suggest the importance of both residues for pentacyclic cyclization by BfOSC3.…”

Section: Resultsmentioning

confidence: 97%

“…These results suggest the importance of both residues for pentacyclic cyclization by BfOSC3. Similar results have been reported previously for SAD1 (β‐amyrin synthase from Avena strigosa ), which transformed enzyme activity (from mainly producing pentacyclic products to mainly producing tetracyclic products) via a single mutation at S728F (Salmon et al ., ).…”

Section: Resultsmentioning

confidence: 97%

Identification of oxidosqualene cyclases from the medicinal legume tree Bauhinia forficata: a step toward discovering preponderant α‐amyrin‐producing activity

et al. 2019

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Summary Triterpenoids are widely distributed among plants of the legume family. However, most studies have focused on triterpenoids and their biosynthetic enzymes in model legumes. We evaluated the triterpenoid aglycones profile of the medicinal legume tree Bauhinia forficata by gas chromatography–mass spectrometry. Through transcriptome analyses, homology‐based cloning, and heterologous expression, we discovered four oxidosqualene cyclases (OSCs) which are responsible for the diversity of triterpenols in B. forficata. We also investigated the effects of the unique motif TLCYCR on α‐amyrin synthase activity. B. forficata highly accumulated α‐amyrin. We discovered an OSC with a preponderant α‐amyrin‐producing activity, which accounted for at least 95% of the total triterpenols. We also discovered three other functional OSCs (BfOSC1, BfOSC2, and BfOSC4) that produce β‐amyrin, germanicol, and cycloartenol. Furthermore, by replacing the unique motif TLCYCR from BfOSC3 with the MWCYCR motif, we altered the function of BfOSC3 such that it no longer produced α‐amyrin. Our results provide new insights into OSC cyclization, which is responsible for the diversity of triterpenoid metabolites in B. forficata, a non‐model legume plant.

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“…Recently, another study describing mutants of the β-amyrin synthase SAD1 from Avena strigosa identified that the mutation Cys563Tyr block initiation of cyclization48. This is the third residue involved with initiation of cyclization so far, since both catalytic aspartic acid and the highly conserved cysteine of the DCTAE motif have previously been demonstrated to be essential for enzymatic activity4950.…”

Section: Resultsmentioning

confidence: 99%

“…Using protein modelling and docking analysis, as performed herein, that work predicted that, in the SAD1 β-amyrin synthase Ser728Phe mutant, the large aromatic side chain of phenylalanine in position 728 interferes with the Phe725, of the catalytic centre, resulting in an interruption of ring expansion of the dammarenyl cation and leading to the formation of truncated tetracyclic oxidosqualene cyclization products. Moreover, the SAD1-Ser728Phe and AtLUP1-Thr729Phe mutants preferentially accepts dioxidosqualene as a substrate when expressed in yeast and cyclizes this to epoxydammarane derivatives, which suggests that Ser728 also affects the ability of the possible OSC substrates to access the active site48.…”

Section: Resultsmentioning

confidence: 99%

Friedelin Synthase from Maytenus ilicifolia: Leucine 482 Plays an Essential Role in the Production of the Most Rearranged Pentacyclic Triterpene

Souza-Moreira

Alves

Pinheiro

et al. 2016

Sci Rep

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Among the biologically active triterpenes, friedelin has the most-rearranged structure produced by the oxidosqualene cyclases and is the only one containing a cetonic group. In this study, we cloned and functionally characterized friedelin synthase and one cycloartenol synthase from Maytenus ilicifolia (Celastraceae). The complete coding sequences of these 2 genes were cloned from leaf mRNA, and their functions were characterized by heterologous expression in yeast. The cycloartenol synthase sequence is very similar to other known OSCs of this type (approximately 80% identity), although the M. ilicifolia friedelin synthase amino acid sequence is more related to β-amyrin synthases (65–74% identity), which is similar to the friedelin synthase cloned from Kalanchoe daigremontiana. Multiple sequence alignments demonstrated the presence of a leucine residue two positions upstream of the friedelin synthase Asp-Cys-Thr-Ala-Glu (DCTAE) active site motif, while the vast majority of OSCs identified so far have a valine or isoleucine residue at the same position. The substitution of the leucine residue with valine, threonine or isoleucine in M. ilicifolia friedelin synthase interfered with substrate recognition and lead to the production of different pentacyclic triterpenes. Hence, our data indicate a key role for the leucine residue in the structure and function of this oxidosqualene cyclase.

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A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases

Cited by 55 publications

References 57 publications

Identification of key amino acid residues determining product specificity of 2,3‐oxidosqualene cyclase in Oryza species

Identification of key amino acid residues determining product specificity of 2,3‐oxidosqualene cyclase in Oryza species

Identification of oxidosqualene cyclases from the medicinal legume tree Bauhinia forficata: a step toward discovering preponderant α‐amyrin‐producing activity

Friedelin Synthase from Maytenus ilicifolia: Leucine 482 Plays an Essential Role in the Production of the Most Rearranged Pentacyclic Triterpene

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