β-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

Abstract

β-Amyrin, a triterpene, is widely distributed in plants and its glycosides confer important biological activities. Mutagenesis studies on β-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 β-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 β-amyrin and determined the chemical structures. The Gly and Ala mutants produced significantly larger amounts of the bicyclic products and a decreased amount of β-amyrin, indicating that the F474 residue was located near the B-ring formation site. Surprisingly, the Ala variant produced (9βH)-polypoda-7,13,17,21-tetraen-3β-ol and (9βH)-polypoda-8(26),13,17,21-tetraen-3β-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 β-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.