Studies on NAD synthetic pathway of hyperthermophile based on genome information

通过基因组测序，在厌氧嗜热古细菌平谷热球菌OT-3中鉴定了一个编码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该编码基因，并对产物进行了纯化。对该蛋白的鉴定表明，该蛋白是迄今为止检测到的最耐热的L-天冬氨酸氧化酶。除了酶的活性外，该酶还在人工电子受体如吩嗪甲硫酸盐、2，6-二氯苯酚-吲哚和铁氰化铁存在下催化L-天冬氨酸脱氢。在原核生物中，LAO是NAD生物合成途径中的第一个酶。通过在公共数据库中进行相似性搜索，在P.Horikoshi OT-3基因组中确定了编码参与该途径的所有其他酶的同源物的基因。编码烟酰胺单核苷酸腺苷转移酶(NMNAT)同源物的基因在大肠杆菌中过表达，所产生的酶被惩罚为同源性。对该酶的表征表明，该酶是一种非常耐热的NMNAT。用高效液相色谱法检测腺苷供体的特异性。在70℃时，三磷酸腺苷是一个重要的捐赠者。然而，在80℃以上，当反应混合物中的腺苷二磷酸或腺苷取代三磷酸腺苷时，检测到相对较小的但显著的NMNAT活性。到目前为止，还没有发现利用ADP或AMP作为腺基供体的NMNAT。目前的研究提供了有趣的信息，其中二磷酸或一磷酸核苷酸可以被腺基转移酶在高温下利用。这些结果表明，P.Horikoshi OT-3在厌氧条件下具有从头合成NAD的途径。我们还在嗜热脂肪芽孢杆菌中发现了一种高度稳定的NAD合成酶。这些酶的X射线结晶学分析正在进行中。

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.