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Analyzing of RuBisCO from the extremely halophilic archaeon for applications

极嗜盐古菌中RuBisCO的分析及其应用

基本信息

批准号：
10460043
负责人：
YOKOTA Akiho
金额：
$ 7.74万
依托单位：
NARA INSTITUTE OF SCIENCE AND TECHNOLOGY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

项目摘要

We performed to clone the gene for RuBisCO from the extremely halophilic Archaeon, Haloferax volcanii in order to analyze the molecular mechanism for salt-resistance of this RuBisCO. The gene for RuBisCO of H. volcanii was higher similar to other Archaeal RuBisCO than that of photosynthetic organisms. Archaeal RuBisCO is called RuBisCO-like protein (RLP) and predicted that this protein does not catalyze the CO_2 fixing reaction in the Calvin cycle because Archaea does not possess the ability of photostnthesis. Non-photosynthetic bacteria including Bacillus subtilis also possess genes for RLP. The function of RLP in non-photosynthetic bacteria and Archaea is unknown yet. The gene for the RLP of B. subtilis is encoded by ylaW, which lies at the first position in the ykrWXYZ operon, close to the ykr7S operon. It was reported that the ykrWXYZ and ykrTS operons were involved in the methionine salvage pathway, but enzymes and catalyzing steps in the methionine salvage pathway of B. subtilis … More are unknown. The reaction steps and enzymes in this pathway were identified to elucidate the catalyzing reaction of RLP by 1H-NMR and UV-visible spectroscopy using recombinant proteins from those operons. This analysis revealed that RLP catalyzes the 2,3-diketo-5-methylthiopentyl-l-phosphate enolase reaction in the fourth step in the methionine salvage pathway. Furthermore, we identified that YkrS is methylthioribose-l-phosphate isomerase, YkrY is methylthioribulose-1-phosphate dehydratase, YkrX is 2-hydmxy-3-keto-5-methylthiopentenyl-1-phosphate phosphatase and YkrZ is 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase in this pathway. Wild type B. subtilis grew on methylthioadenosine (MTA), an intermediate in the methionine salvage pathway, as the sole sulfur compound. A mutant with the RLP gene disrupted did not grow on MTA. Interestingly, the mutant was rescued for growth on MTA by introducing the gene of photosynthetic RuBisCO from a photosynthetic bacterium. This result indicates that photosynthetic RuBisCO also catalyzes the same reaction step as RLP. Less

为了分析该RuBisCO耐盐的分子机制，我们从极端嗜盐古菌——火山盐铁古菌中克隆了RuBisCO基因。H. volcanii的RuBisCO基因与其他古生菌的RuBisCO基因相似度高于光合生物。古细菌RuBisCO被称为RuBisCO样蛋白（RLP），并预测该蛋白在卡尔文循环中不催化CO_2固定反应，因为古细菌不具有光合作用的能力。包括枯草芽孢杆菌在内的非光合细菌也具有RLP基因。RLP在非光合细菌和古细菌中的作用尚不清楚。枯草芽孢杆菌RLP基因由ylaW编码，位于ykrWXYZ操纵子的第一个位置，靠近ykr7S操纵子。据报道，ykrWXYZ和ykrTS操纵子参与了枯草芽孢杆菌的蛋氨酸回收途径，但更多的酶和催化步骤尚不清楚。利用这些操纵子的重组蛋白，通过1H-NMR和uv -可见光谱分析，鉴定了该途径中的反应步骤和酶，并阐明了RLP的催化反应。分析表明，RLP在蛋氨酸回收途径的第四步催化2,3-二酮-5-甲基硫喷戊基-磷酸烯醇化酶反应。此外，我们在该途径中鉴定出YkrS为甲基硫核糖-1-磷酸异构酶，YkrY为甲基硫核糖-1-磷酸脱水酶，YkrX为2-羟基-3-酮-5-甲基硫喷戊基-1-磷酸磷酸酶，YkrZ为1,2-二羟基-3-酮-5-甲基硫喷戊烯双加氧酶。野生型枯草芽孢杆菌以甲基硫腺苷（MTA）为唯一的硫化合物生长，甲基硫腺苷是蛋氨酸回收途径的中间体。RLP基因被破坏的突变体没有在MTA上生长。有趣的是，该突变体通过引入来自光合细菌的光合RuBisCO基因而得以在MTA上生长。这一结果表明光合RuBisCO也催化了与RLP相同的反应步骤。少