Mohan R S, Yee N K, Coates R M, Ren Y Y, Stamenkovic P, Mendez I, West C A
Department of Chemistry, University of Illinois, Urbana 61801, USA.
Arch Biochem Biophys. 1996 Jun 1;330(1):33-47. doi: 10.1006/abbi.1996.0223.
The biosynthesis of diterpene hydrocarbons with enzyme extracts from rice cell suspension cultures was investigated to verify proposed pathways and intermediates in the production of the momilactone and oryzalexin phytoalexins. Diterpene synthase activity in cells treated with chitin to elicit the phytoalexin response was compared with the activity in untreated cells using the acyclic substrates 1-3H- and [1-3H] (E,Z,E)-geranylgeranyl diphosphates (GGPPs 4-OPP and 11-OPP) as well as the bicyclic substrates [15-3H]ent-copalyl and [15-3H] syn-copalyl diphosphates (CPPs, 5-OPP, and 6-OPP). ent-kaurene (7), ent-sanda, racopimaradiene (8), 9 beta H-pimara-7,15-diene (9), and stemar-13-ene (10) were identified as major products by comparisons with authentic standards. Marked increases in diterpene synthase activities were observed with enzyme from chitin-treated cells: (E,E,E)-GGPP (approximately 100 fold), ent-CPP (approximately 3 fold), and syn-CPP (approximately 60 fold). The very low conversions of (E,Z,E)-GGPP to hydrocarbon products excludes its role in the biosynthesis of 9,10-syn-diterpenes in rice cells. ent-Kaurene was the major diterpene formed from ent-CPP with enzyme from unelicited cells. In contrast the enzyme from chitin-treated cells converted ent-CPP to a mixture of ent-kaurene, ent-sandaracopimaradiene, and a third unidentified diterpene. With syn-CPP as substrate the induced syntheses afforded a mixture of 9 beta-pimaradiene, stemarene, and a third, unidentified syn-diterpene. Overall the results are consistent with the hypothesis that rice cells respond to treatment with chitin fragments by producing new diterpene synthases not present in the untreated cells. These induced cyclases initiate phytoalexin biosynthesis by diverting (E,E,E)-GGPP into new cyclization modes that produce ent-sandaracopimaradiene, stemarene, and 9 beta-pimaradiene, the presumed precursors to oryzalexins A-F, oryzalexin S, and momilactones A-C, respectively. The intermediate role of 9,10-syn-CPP in syn diterpene biosynthesis is verified.
研究了利用水稻细胞悬浮培养物中的酶提取物进行二萜烃生物合成，以验证在稻瘟菌素和水稻抗毒素植保素生产中提出的途径和中间体。使用无环底物1-³H-和1-³H-香叶基香叶基二磷酸（GGPPs 4-OPP和11-OPP）以及双环底物[15-³H]内贝壳杉烯基和[15-³H]顺式贝壳杉烯基二磷酸（CPPs，5-OPP和6-OPP），比较了用几丁质处理以引发植保素反应的细胞中的二萜合酶活性与未处理细胞中的活性。通过与标准品比较，确定内贝壳杉烯（7）、内贝壳杉二醇、消旋海松二烯（8）、9βH-海松-7,15-二烯（9）和异海松-13-烯（10）为主要产物。观察到用几丁质处理的细胞的酶提取物中二萜合酶活性显著增加：(E,E,E)-GGPP（约100倍）、内CPP（约3倍）和顺式CPP（约60倍）。(E,Z,E)-GGPP向烃类产物的转化率非常低，排除了其在水稻细胞中9,10-顺式二萜生物合成中的作用。内贝壳杉烯是未诱导细胞的酶提取物将内CPP转化形成的主要二萜。相比之下，用几丁质处理的细胞的酶提取物将内CPP转化为内贝壳杉烯、内贝壳杉海松二烯和第三种未鉴定二萜的混合物。以顺式CPP为底物时，诱导合成产生了9β-海松二烯、异海松烯和第三种未鉴定的顺式二萜的混合物。总体而言，结果与以下假设一致：水稻细胞通过产生未处理细胞中不存在的新二萜合酶来响应几丁质片段处理。这些诱导的环化酶通过将(E,E,E)-GGPP转移到新的环化模式来启动植保素生物合成，这些模式分别产生内贝壳杉海松二烯、异海松烯和9β-海松二烯，它们分别是水稻抗毒素A-F、水稻抗毒素S和稻瘟菌素A-C的假定前体。验证了9,10-顺式CPP在顺式二萜生物合成中的中间作用。
