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Studies on NAD synthetic pathway of hyperthermophile based on genome information

基于基因组信息的超嗜热菌NAD合成途径研究

基本信息

批准号：
13680716
负责人：
SAKURABA Haruhiko
金额：
$ 2.3万
依托单位：
The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
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/grant/KAKENHI-PROJECT-13680716/
关键词：
Hyperthermophile Pyrococcus horikoshii Archaea NAD biosynthesis Genome information L-Aspartate oxidase X-ray crystalographic analysis NAD synthase Pyrococcus horiloshii NAD X線結晶構造解析 L-アスパラギン酸脱水素酵素

项目摘要

A gene encoding the L-aspartate oxidase (LAO) homologue was identified via genome sequencing in the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3. We succeeded in expressing the encoding gene in Escherichia coil and purified the product to homogeneity. Characterization of the protein revealed that it was the most thermostable L-aspartate oxidase detected so far. In addition to the oxidase activity, the enzyme catalyzed L-aspartate dehydrogenation in the presence of an artificial electron acceptor such as phenazine methosulfate, 2,6-dichlorophenol-indophenol and ferricyanide. LAO is known to function as the first enzyme in the de novo NAD biosynthetic pathway in prokaryotes. By a similarity search in public databases, the genes that encode the homologue of all other enzymes involved in the pathway were identified in the P. horikoshi OT-3 genome. A gene encoding nicotinamide mononucleotide adenylytransferase (NMNAT) homologue was overexpressed in E. coli, and the produced enzyme was punfied to homogeneity. Characterization of the enzyme revealed that it is an extremely thermostable NMNAT. The adenylyl group donor specificity was examined by high-performance liquid chromatography. At 70℃, ATP was a prominent donor. However, above 80℃, a relatively small, but significant NMNAT activity was detected when ATP was replaced by ADP or AMP in the reaction mixture. To date, NMNAT that utilizes ADP or AMP as an adenylyl group donor has not been found . The present study provides interesting information in which di-or mono-phosphate nucleotide can be utilized by adenylyltransferase at high temperature. These results suggest that P. horikoshi OT-3 has the de novo NAD biosynthetic pathway under anaerobic conditions. We also discovered a highly stable NAD synthase in Bacillus stearothermophilus. X-ray crystalographic analyses of these enzymes are in progress.

通过基因组测序在厌氧超嗜热古菌Pyrococcus horikoshii OT-3中鉴定了编码L-天冬氨酸氧化酶（LAO）同源物的基因。我们成功地在大肠杆菌中表达了编码基因，并纯化了产物。蛋白质的表征表明，它是迄今为止检测到的最耐热的L-天冬氨酸氧化酶。除了氧化酶的活性，酶催化L-天冬氨酸脱氢的人工电子受体，如吩嗪硫酸甲酯，2，6-二氯苯酚-靛酚和铁氰化物的存在下。已知LAO在原核生物中作为NAD从头生物合成途径中的第一个酶起作用。通过在公共数据库中的相似性搜索，在堀越拟杆菌OT-3基因组中鉴定了编码参与该途径的所有其他酶的同源物的基因。将编码烟酰胺单核苷酸腺苷酰转移酶（NMNAT）同源物的基因在E.大肠杆菌中，并将所产生的酶纯化至均一。该酶的表征表明，它是一种非常热稳定的NMNAT。通过高效液相色谱法检查腺苷酰基团供体特异性。在70℃时，ATP是主要的供体。而在80℃以上，当ATP被ADP或AMP取代时，则可检测到相对较小但显著的NMNAT活性。迄今为止，还没有发现利用ADP或AMP作为腺苷酰基供体的NMNAT。本研究为腺苷酸转移酶在高温下利用二磷酸或单磷酸核苷酸提供了有趣的信息。这些结果表明，P. horikoshi OT-3在厌氧条件下具有从头NAD生物合成途径。我们还在嗜热脂肪芽孢杆菌中发现了一种高度稳定的NAD合酶。这些酶的X射线晶体学分析正在进行中。