A second novel dye-linked L-proline dehydrogenase complex is present in the hyperthermophilic archaeon Pyrococcus horikoshii OT-3
A second novel dye-linked L-proline dehydrogenase complex is present in the hyperthermophilic archaeon Pyrococcus horikoshii OT-3
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DOI：
10.1111/j.1742-4658.2005.04810.x
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发表时间：
2005-08-01
期刊：
影响因子：
5.4
通讯作者：
Ohshima, T
中科院分区：
文献类型：
--
作者：
Kawakami, R;Sakuraba, H;Ohshima, T
当超嗜热古菌Pyrococcus horikoshii OT-3的粗提物在聚丙烯酰胺凝胶上电泳时，检测到染料连接的L-脯氨酸脱氢酶（PDH 1和PDH 2）的两个可区分的活性条带。经纯化后，发现PDH 1由分子量为56和43 kDa的两个不同亚基组成，而PDH 2由分子量为52、46、20和8 kDa的四个不同亚基组成。PDH 1和PDH 2的天然分子量分别为440和101 kDa，表明PDH 1具有α（4）β（4）结构，而PDH 2具有α（4）β（4）结构。发现PDH 2类似于来自Thermococcus profundus的染料连接的L-脯氨酸脱氢酶复合物，但PDH 1是不同类型的酶。在大肠杆菌中产生该酶后，高效液相色谱显示PDH 1复合物含有黄素FMN和FAD以及ATP。每个亚基的基因表达和生化分析表明，β亚基结合FAD和表现出脯氨酸脱氢酶活性，而α亚基结合ATP，但不像T.深酶，它既不表现出脯氨酸脱氢酶，也不表现出NADH脱氢酶活性。FMN不与任何一个亚基结合，表明它位于α和β亚基之间的界面。氨基酸序列的比较表明，ADP结合基序在α亚基的PDH 1明显不同于α亚基的PDH 2。因此，看来第二个新的染料连接的L-脯氨酸脱氢酶复合物中产生的P. horikoshii。
Two distinguishable activity bands for dye-linked L-proline dehydrogenase (PDH1 and PDH2) were detected when crude extract of the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 was run on a polyacrylamide gel. After purification, PDH1 was found to be composed of two different subunits with molecular masses of 56 and 43 kDa, whereas PDH2 was composed of four different subunits with molecular masses of 52, 46, 20 and 8 kDa. The native molecular masses of PDH1 and PDH2 were 440 and 101 kDa, respectively, indicating that PDH1 has an alpha(4)beta(4) structure, while PDH2 has an alpha(4)beta(4) structure. PDH2 was found to be similar to the dye-linked L-proline dehydrogenase complex from Thermococcus profundus, but PDH1 is a different type of enzyme. After production of the enzyme in Escherichia coli, high-performance liquid chromatography showed the PDH1 complex to contain the flavins FMN and FAD as well as ATP. Gene expression and biochemical analyses of each subunit revealed that the beta subunit bound FAD and exhibited proline dehydrogenase activity, while the alpha subunit bound ATP, but unlike the corresponding subunit in the T. profundus enzyme, it exhibited neither proline dehydrogenase nor NADH dehydrogenase activity. FMN was not bound to either subunit, suggesting it is situated at the interface between the alpha and beta subunits. A comparison of the amino-acid sequences showed that the ADP-binding motif in the alpha subunit of PDH1 clearly differs from that in the alpha subunit of PDH2. It thus appears that a second novel dye-linked L-proline dehydrogenase complex is produced in P. horikoshii.