The genetic and biochemical research of lipid biosynthesis in archaea

古细菌脂质生物合成的遗传和生化研究

• 批准号: 13450338
• 负责人: NISHINO Tokuzo
• 金额: $ 9.92万
• 依托单位: TOHOKU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13450338/
• 关键词: Archaea; isoprenoid; prenyl tronsferase; prenyl quinone; molecular evolution; carotenoid; 糖キャリア脂質

1. We Succeeded in isolating the gene of hexaprenyl diphosphate synthase, which is considered to produce the precursor of the C30 side-chain of caldariellaquinone, from a thermoacidophilic archaeon Sulfolobus solfataticus based on homology searching from its whole-genomesequence. The gene was exogenously expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. In consequence, it was proved to be the homomultimeric-type enzyme, which deffers from well-studied bacterial mudium-chain prenyl diphosphate synthases that have heterodimeric structures. Moreover, as the result of the phylogenetic analysis of the sequences of various prenyl diphosphate synthases, the archaeal enzyme was suggested to be closely related with eukaryotic short-chain enzymes, not with other medium-and long-chain enzymes; This idea was also supported by the result of a mutagenic study, in which partical sequences of prenyl diphosphate synthases from other organisms were site- derectedl … More y introduced in the srchaeal hexaprenyl diphosphate synthase.2. The gene of isopentenyl diphosphate isomerase was isolated from Sulfolobus shibatae and expressed in E. coli. This enzyme is important for biosynthesis of isoprenoid compounds in archaea because it catalyzes the first step of their biosynthetic pathways. The enzyme was proved to have novel properties; for axample, it shows co-enzyme requirement largely different with those of known isopentenyl hiphosphate synthases. Besides, the enzyme was suggested to have structural similarity with some types of oxidoreductases.3. We isolated the genes of various enzymes that catalyze the reactions of isoprenoid biosynthesis and proved their functions by expressing them in the cells of E. coli. The enzyme are thought to be valuable to investigate the evolutional route of each group of enzyes in which they are contained, because they have uniwue characteristics. For example, lycopene cyclase from S. solfataricus was proved to have the fusion-type structure specific for archaeal ones. Less

1. 我们通过全基因组序列的同源性搜索，成功从嗜热嗜酸古菌硫磺硫菌中分离出六异戊二烯二磷酸合酶基因，该基因被认为是产生卡达里埃醌C30侧链的前体。该基因在大肠杆菌中外源表达，并对重组酶进行纯化和表征。结果，它被证明是同多聚体型酶，与经过充分研究的具有异二聚体结构的细菌泥链异戊二烯基二磷酸合酶不同。此外，对各种异戊二烯基二磷酸合酶序列进行系统发育分析的结果表明，该古菌酶与真核短链酶密切相关，而不是与其他中链和长链酶​​密切相关；这一想法也得到了诱变研究的结果的支持，其中来自其他生物体的异戊二烯基二磷酸合酶的部分序列被定点引入到现有的己烯基二磷酸合酶中。2。从柴氏硫化叶菌中分离出异戊烯基二磷酸异构酶基因，并在大肠杆菌中表达。这种酶对于古细菌中类异戊二烯化合物的生物合成很重要，因为它催化其生物合成途径的第一步。该酶被证明具有新颖的特性；例如，它显示出与已知的异戊烯基磷酸合酶的辅酶需求有很大不同。此外，该酶与某些类型的氧化还原酶具有结构相似性。 3．我们分离了催化类异戊二烯生物合成反应的各种酶的基因，并通过在大肠杆菌细胞中表达它们证明了它们的功能。该酶被认为对于研究它们所包含的每组酶的进化路线很有价值，因为它们具有独特的特征。例如，来自S. solfataricus的番茄红素环化酶被证明具有古菌特有的融合型结构。较少的

项目成果

M.Nagaki et al.: "Substrate specificity of thermostable farnesyl diphosphate synthase with respect to 4-alkyl group homologs of isopentenyl diphosphate"J. Mol. Catal. B: Enzym. 17. 81-89 (2002)

M.Nagaki 等人：“热稳定性法尼基二磷酸合酶对异戊烯基二磷酸的 4-烷基同系物的底物特异性”J。

Y. Maki, M. Nagaki, T. Nishino, and T. Koyama: "Usefulness of prenyltransferase for organic synthesis: Carbon-carbon bond forming reactions by prenyltransferases and their mutated enzymes"J. Syn. Org. Chem. Japan. 60. 783-793 (2002)

Y. Maki、M. Nagaki、T. Nishino 和 T. Koyama：“异戊二烯基转移酶在有机合成中的用途：异戊二烯基转移酶及其突变酶的碳-碳键形成反应”J。

M.Nagaki et al.: "Artificial substrates of medium-chain elongation enzymes, hexaprenyl-and heptaprenyl diphosphate synthases"Bioorg. Med. Chem. Lett.. 11. 2157-2159 (2001)

M.Nagaki 等人：“中链延伸酶、己烯基和庚烯基二磷酸合酶的人工底物”Bioorg。

E-i.Fukusaki et al.: "Introduction of the archaebacterial geranylgeranyl pyrophosphate synthase gene into Clamydomonas reinhardtii chloroplast"J. Biosci. Bioeng.. 95. 283-287 (2003)

E-i.Fukusaki 等：“将古细菌香叶基香叶基焦磷酸合酶基因引入莱茵衣藻叶绿体”J.

M. Nagaki, H. Yamamoto, A. Takahashi, Y. Maki, J. Itabashi, T. Nishino, and T. Koyama: "Substrate specificity od thermostable famesyl diphosphate synthase with respect to 4-alkyl group homologs of isopentenyl diphosphate"J. Mol. Catal. B: Enzym.. 17. 81-8

M. Nagaki、H. Yamamoto、A. Takahashi、Y. Maki、J. Itabashi、T. Nishino 和 T. Koyama：“热稳定法呢基二磷酸合酶对于异戊烯基二磷酸的 4-烷基同系物的底物特异性”J