Molecular and functional characterization of D-3-phosphoglycerate dehydrogenase in the serine biosynthetic pathway of the hyperthermophilic archaeon Sulfolobus tokodaii
Molecular and functional characterization of D-3-phosphoglycerate dehydrogenase in the serine biosynthetic pathway of the hyperthermophilic archaeon Sulfolobus tokodaii
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DOI：
10.1016/j.abb.2007.11.010
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发表时间：
2008-02-15
影响因子：
3.9
通讯作者：
Ohshima, Toshihisa
中科院分区：
文献类型：
--
作者：
Shimizu, Yasuhiro;Sakuraba, Haruhiko;Ohshima, Toshihisa
通过筛选可能有助于超嗜热古菌中L-丝氨酸生物合成的酶的数据库，在东京硫化叶菌菌株7的基因组中发现编码D-3-磷酸甘油酸脱氢酶（PGDH; EC 1.1.1.95）同源物的基因（ST 1218）。在大肠杆菌中表达该基因后，对重组酶的PGDH活性进行了评估。使用常规色谱步骤获得均质PGDH，尽管在纯化期间，如果酶储存在塑料管中而不是玻璃管中，则观察到酶活性的意外下降。纯化的酶是一个同源二聚体的亚基分子量约为35 kDa，是高度热稳定的。它优选地充当NAD依赖性D-3-磷酸甘油酸（3 PGA）脱氢酶。虽然NADP没有电子受体活性，但NADPH和NADH都充当电子供体。动力学分析表明，酶反应通过Theorell-Chance Bi-Bi机制进行。与大肠杆菌PGDH不同，S. tokodaii酶不被L-丝氨酸抑制。此外，磷酸根和硫酸根离子增强了NAD依赖的3 PGA氧化和逆反应，而NADPH依赖的3-磷酸羟基丙酮酸（PHP）还原被抑制。因此S. tokodaii PGDH似乎受到一种在其他地方没有看到的新的调节机制的影响。数据库分析表明，ST 1218基因与ST 1217基因形成一个簇，并在大肠杆菌中表达的ST 1217产物的功能分析表明，它具有L-谷氨酸-PHP转氨酶活性。综上所述，我们的研究结果代表了第一个例子磷酸化丝氨酸途径中的超嗜热古菌。(c)2007年爱思唯尔公司All rights reserved.
A gene (ST1218) encoding a D-3-phosphoglycerate dehydrogenase (PGDH; EC 1.1.1.95) homolog was found in the genome of Sulfolobus tokodaii strain 7 by screening a database of enzymes likely to contribute to L-serine biosynthesis in hyperthermophilic archaea. After expressing the gene in Escherichia coli, the PGDH activity of the recombinant enzyme was assessed. Homogeneous PGDH was obtained using conventional chromatography steps, though during the purification an unexpected decline in enzyme activity was observed if the enzyme was stored in plastic tubes, but not in glass ones. The purified enzyme was a homodimer with a subunit molecular mass of about 35 kDa and was highly thermostable. It preferably acted as an NAD-dependent D-3-phosphoglycerate (3PGA) dehydrogenase. Although NADP had no activity as the electron acceptor, both NADPH and NADH acted as electron donors. Kinetic analyses indicated that the enzyme reaction proceeds via a Theorell-Chance Bi-Bi mechanism. Unlike E coli PGDH, the S. tokodaii enzyme was not inhibited by L-serine. In addition, both the NAD-dependent 3PGA oxidation and the reverse reaction were enhanced by phosphate and sulfate ions, while NADPH-dependent 3-phosphohydroxypyruvate (PHP) reduction was inhibited. Thus S. tokodaii PGDH appears to be subject to a novel regulatory mechanism not seen elsewhere. A database analysis showed that ST 1218 gene forms a cluster with ST 1217 gene, and a functional analysis of the ST 1217 product expressed in E coli revealed that it possesses L-glutamate-PHP aminotransferase activity. Taken together, our findings represent the first example of a phosphorylated serine pathway in a hyperthermophilic archaeon. (c) 2007 Elsevier Inc. All rights reserved.