In this study, mono- and pentacyclic C(35) terpenes from Bacillus subtilis were biosynthesized via the cyclization of C(35) isoprenoid using purified enzymes, including the first identified new terpene cyclase that shows no sequence homology to any of the known terpene cyclases. On the basis of these findings, we propose that these C(35) terpenes should be called the new family of "sesquarterpenes."

This study demonstrates that a tetraprenyl-β-curcumene cyclase, which was originally identified as a sesquarterpene cyclase that converts a head-to-tail type of monocycle to a pentacycle, also cyclizes a tail-to-tail type of linear squalene into a bicyclic triterpenol, 8α-hydroxypolypoda-13,17,21-triene. The 8α-hydroxypolypoda-13,17,21-triene was found to be a natural triterpene from B. megaterium. It was also demonstrated that cyclizations of both tetraprenyl-β-curcumene and squalene occurred with a purified B. megaterium TC homologue in the same reaction mixture. These results suggest that the tetraprenyl-β-curcumene cyclase is bifunctional, cyclizing both tetraprenyl-β-curcumene and squalene in vivo. This is the first report describing a bifunctional terpene cyclase, which biosynthesizes two classes of cyclic terpenes with different numbers of carbons as natural products in the organism.

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The acyclic molecule squalene (1) is cyclized into 6,6,6,6,5-fused pentacyclic hopene (2) and hopanol (3; ca. 5:1) through the action of Alicyclobacillus acidocaldarius squalene-hopene cyclase (AaSHC). The polycyclization reaction proceeds with regio- and stereochemical specificity under precise enzymatic control. This pentacyclic hopane skeleton is generated by folding 1 into an all-chair conformation. The Ala306 residue in AaSHC is conserved in known squalene-hopene cyclases (SHCs); however, increasing the steric bulk (A306T and A306V) led to the accumulation of 6,6,6,5-fused tetracyclic scaffolds possessing 20R stereochemistry in high yield (94 % for A306V). The production of the 20R configuration indicated that 1 had been folded in a chair-chair-chair-boat conformation; in contrast, the normal chair-chair-chair-chair conformation affords the tetracycle with 20S stereochemistry, but the yield produced by the A306V mutant was very low (6 %). Consequently, bulk at position 306 significantly affects the stereochemical fate during the polycyclization reaction. The SHC also accepts (3R) and (3S)-2,3-oxidosqualenes (OXSQs) to generate 3α,β-hydroxyhopenes and 3α,β-hydroxyhopanols through polycyclization initiated at the epoxide ring. However, the Val and Thr mutants generated epoxydammarane scaffolds from (3R)-OXSQ; this indicated that the polycyclization cascade started in these instances at the terminal double bond position. This work is the first to report the polycyclization of oxidosqualene starting at the terminal double bond.

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