30(C-6) 活性中心探索プローブを用いたトリテルペン合成酵素反応機構の解析(口頭発表の部)

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1. 1-Methylidenesqualene (1-MS) and 25-MS were converted to 30-methylidene-hop-22(29)-ene by recombinant squalene: hopene cyclase from Alicyclobacillus acidocaldarius. It was remarkable that both analogues afforded the same product; the hopanyl intermediate cation, stabilized by the methylidene residue enabled a rotation of the isobutenyl group at C-21 prior to the final proton elimination. Further, the enzyme also converted 26-MS and 27-MS to novel pentacyclic C_<31> polyprenoids; a dammarene derivative with a 6.6.6.5+6 ring system and 26-methylidene-hop-22(29)-ene, respectively. The broad substrate specificity of the enzyme provided important information on the structure and function of squalene: hopene cyclase. 2. Recombinant oxidosqualene: β-amyrin cyclase from Pisum sativum converted 22,23-dihydro-2,3-oxidosqualene into a 4: 1 mixture of euph-7-en-3β-ol and bacchar-12-en-3β-ol. This is the first demonstration of the enzymatic formation of the baccharene skeleton with a six-membered D-ring. In the absence of the terminal double bond, the proton initiated cyclization first generated the tetracyclic dammarenyl cation, followed by a backbone rearrangement with loss of H-7α leading to the formation of euph-7-en-3β-ol, while D-ring expansion to the baccharenyl cation and subsequent 1,2-hydride shifts with H-12α elimination yielded bacchar-12-en-3β-ol. The recombinant enzyme also converted 24,30-bisnor-2,3-oxidosqualene into a 3: 1: 0.2 mixture of 29,30-bisnor-β-amyrin, 29,30-bisnorgermanicol, and 29,30-bisnor-δ-amyrin. Furthermore, incubation with [23-<13>^C]- and [23,23-2^H]-labeled isotopomers established that the cyclization reaction proceeded through formation of a thermodynamically favored bisnoroleanyl secondary cation with a less-strained six-membered E-ring.
天然有機化合物討論会の論文
2004-10-01

30(C-6) 活性中心探索プローブを用いたトリテルペン合成酵素反応機構の解析(口頭発表の部)

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