Direct formation of the sesquiterpeonid ether liguloxide by a terpene synthase in Senecio scandens

Isotopic Labeling Experiments Solve the Hedycaryol Problem

“…We aimed at the usage of two hedycaryol synthases, one from a plant and one from a bacterium (Figure S1), as plant and bacterial enzymes usually make different enantiomers. ,, PtTPS5 from P. trichocarpa was considered to be a good starting point because for tobacco 5- epi -aristolochene synthase (TEAS), the exchange of the active site residue Y520 with Phe (Y520F) has been demonstrated to interrupt the cyclization cascade at the intermediate 5 , suggesting its involvement in the reprotonation for the second cyclization to 5- epi -aristolochene . Also, for other 1,10-cyclizing plant terpene synthases that produce eudesmane or guaiane sesquiterpene hydrocarbons or alcohols, the accumulation of intermediate 1 or 5 , respectively, was observed upon the exchange of the corresponding Tyr residue. − With FPP as the substrate, the enzyme variant Y521F of PtTPS5 also resulted in an abolished production of 8 and 9 with accumulation of 1 , but the production was strongly reduced (5.3 ± 0.3% of wild-type level of 8 + 9 , Figure ); therefore, this mutant was not suitable for labeling experiments. As previously observed for pseudolaratriene synthase (PxaTPS8) from Pseudolarix amabilis , mutation of the first Arg in the conserved RxR motif (R265M) gave an inactive enzyme.…”

“…The enzyme variants E379M and E379Q lost the ability to produce 8 and 9 almost completely, with some retained production of 1 (22 ± 3 and 14 ± 2%, respectively), establishing the importance of this residue that shows a long-range interaction with Mg 2+ in the TEAS structure for catalysis. The observed amino acid residues near the helix G kink in plant type I terpene synthases are diverse, and it has been shown in several mutational studies that exchanges in this region can dramatically influence the product profile. − Specifically, the exchanges T409G in Zea mays eudesmanediol synthase (ZmEDS) and H415A in Senecio scandens liguloxide synthase (SsLOS) caused a product shift toward the intermediate 1 . , The substitutions C403Y and C403L (equal to T409 in ZmEDS) yielded inactive proteins, whereas C403A and C403S lost the ability to form 8 and 9 , with a shift toward 1 in good yields (70 ± 10% and 65 ± 3%, respectively). The exchange Y404F (equal to H415 in SsLOS) showed a moderately reduced activity, with 1 and 8 + 9 produced in nearly equal amounts.…”

“…34−37 Specifically, the exchanges T409G in Zea mays eudesmanediol synthase (ZmEDS) and H415A in Senecio scandens liguloxide synthase (SsLOS) caused a product shift toward the intermediate 1. 30,31 The substitutions C403Y and C403L (equal to T409 in ZmEDS) yielded inactive proteins, whereas C403A and C403S lost the ability to form 8 and 9, with a shift toward 1 in good yields (70 ± 10% and 65 ± 3%, respectively). The exchange Y404F (equal to H415 in SsLOS) showed a moderately reduced activity, with 1 and 8 + 9 produced in nearly equal amounts.…”

Transcriptome Analysis of Antrodia cinnamomea Mycelia from Different Wood Substrates

“…Terpenoids comprised of isoprene (C5) units, and their derivatives and are one of the major secondary metabolites of fungi and are widely distributed in nature [ 13 ]. Terpenoid is primarily synthesized by the C5 precursors, such as isopentenyl diphosphate (IPP) and dimethyl allyl diphosphate (DMAPP) through the mevalonate (MVA) and methyl tetrahydroxyl 4-phosphate (MEP) pathways [ 14 ].…”