Euphorbia tirucalli ß-Amyrin Synthase: Critical Roles of Steric Sizes at Val483 and Met729 and the CH-π Interaction between Val483 and Trp534 for Catalytic Action.

The functions of Val483, Trp534, and Met729 in Euphorbia tirucalli ß-amyrin synthase were revealed by comparing the enzyme activities of site-directed mutants against that of the wild type. The Gly and Ala variants with a smaller bulk size at position 483 predominantly afforded monocyclic camelliol C, which suggested that the orientation of the (3S)-2,3-oxidosqualene substrate was not appropriately arranged in the reaction cavity as a result of the decreased bulk size, leading to failure of its normal folding into the chair-chair-chair-boat-boat conformation. The Ile variant, with a somewhat larger bulk, afforded ß-amyrin as the dominant product. Intriguingly, various variants of Trp534 exhibited significantly decreased enzymatic activities and provided no aberrantly cyclized products, although the aromatic Phe and Tyr residues were incorporated and the steric sizes of the aliphatic residues were altered. Therefore, the Trp534 residue does not stabilize the transient cation through a cation-π interaction. Furthermore, the Trp residue, with the largest steric bulk among all natural amino acids, is essential for high enzymatic activity. Robust CH-π complexation between the Val483 and Trp534 residues is proposed herein. Altering the steric bulk at the Met729 position afforded the pentacyclic skeletons. Thus, Met729 is positioned at the E-ring formation site. More detailed insights into the functions of the Val483, Trp534, and Met729 residues are provided by homology modeling.

Correction: ß-Amyrin synthase from Euphorbia tirucalli L. functional analyses of the highly conserved aromatic residues Phe413, Tyr259 and Trp257 disclose the importance of the appropriate steric bulk, and cation-π and CH-π interactions for the efficient catalytic action of the polyolefin cyclization cascade.

Correction for 'ß-Amyrin synthase from Euphorbia tirucalli L. functional analyses of the highly conserved aromatic residues Phe413, Tyr259 and Trp257 disclose the importance of the appropriate steric bulk, and cation-π and CH-π interactions for the efficient catalytic action of the polyolefin cyclization cascade' by Ryousuke Ito et al., Org. Biomol. Chem., 2017, 15, 177-188.

ß-Amyrin synthase from Euphorbia tirucalli L. functional analyses of the highly conserved aromatic residues Phe413, Tyr259 and Trp257 disclose the importance of the appropriate steric bulk, and cation-π and CH-π interactions for the efficient catalytic action of the polyolefin cyclization cascade.

Many of the functions of the active site residues in ß-amyrin synthase and its catalytic mechanism remain unclear. Herein, we examined the functions of the highly conserved Phe413, Tyr259, and Trp257 residues in the ß-amyrin synthase of Euphorbia tirucalli. The site-specific mutants F413V and F413M [corrected] showed nearly the same enzymatic activities as the wild type, indicating that π-electrons are not needed for the catalytic reaction. However, the F413A [corrected] mutant yielded a large amount of the tetracyclic dammarane skeleton, with decreased production of ß-amyrin. This indicates that the Phe413 [corrected] residue is located near the D-ring formation site and works to position the oxidosqualene substrate correctly within the reaction cavity. On the other hand, the major catalysis-related function of the Tyr259 and Trp257 residues is to yield their π-electrons to the cationic intermediates. The Y259F variant showed nearly equivalent activity to that of the wild type, but aliphatic mutants such as the Ala, Val, and Leu variants showed significantly decreased the activity and yielded the tetracyclic dammarane scaffold, strongly demonstrating that the Tyr259 residue stabilizes the baccharenyl secondary cation via cation-π interaction. The aliphatic variants of Trp257 exhibited remarkably decreased enzymatic activity, and lupeol was produced in a high production ratio, indicating that Trp257 stabilizes the oleanyl cation via cation-π interaction. The aromatic Phe and Tyr mutants exhibited high activities owing to their more increased π-electron density relative to that of the aliphatic mutants, but lupeol was produced in a significantly high yield besides ß-amyrin. The Trp residue is likely to be responsible for the robust binding of Me-30 through CH-π interaction. The decreased π-electron density of the Phe and Tyr mutants compared to that of Trp would have resulted in the high production of lupeol.

ß-Amyrin synthase from Euphorbia tirucalli. Steric bulk, not the π-electrons of Phe, at position 474 has a key role in affording the correct folding of the substrate to complete the normal polycyclization cascade.

ß-Amyrin, a triterpene, is widely distributed in plants and its glycosides confer important biological activities. Mutagenesis studies on ß-amyrin synthase are very limited as compared with those of squalene-hopene cyclase and lanosterol synthase. This study was conducted to elucidate the function of the F474 residue of Euphorbia tirucalli ß-amyrin cyclase, which is highly conserved in the superfamily of oxidosqualene cyclases. Nine site-specific variants with Gly, Ala, Val, Leu, Met, Tyr, Trp, His, and Thr were constructed. We isolated 9 products from these mutants in addition to ß-amyrin and determined the chemical structures. The Gly and Ala mutants produced significantly larger amounts of the bicyclic products and a decreased amount of ß-amyrin, indicating that the F474 residue was located near the B-ring formation site. Surprisingly, the Ala variant produced (9ßH)-polypoda-7,13,17,21-tetraen-3ß-ol and (9ßH)-polypoda-8(26),13,17,21-tetraen-3ß-ol, which are generated from a chair-boat folding conformation. This is the first report describing the conformational change from the chair-chair into the chair-boat folding conformation among the reported mutagenesis studies of oxidosqualene cyclases. Substitution with aliphatic amino acids lacking π-electrons such as Val, Leu, and Met led to a significantly decreased production of bicyclic compounds, and in turn exhibited a higher production of ß-amyrin. Furthermore, the Leu and Met variants exhibited high enzymatic activities: ca. 74% for Leu and ca. 91% for Met variants as compared to the wild-type. These facts unambiguously demonstrate that the major role of Phe474 is not to stabilize the transient cation via cation-π interaction, but is to confer the appropriate steric bulk near the B-ring formation site, leading to the completion of the normal polycyclization pathway without accumulation of abortive cyclization products.