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Application of Hyperthennophilir 2-Deoxy-D-Riltose-5-Phosphate Aldolase

嗜热菌2-脱氧-D-利托糖-5-磷酸醛缩酶的应用

基本信息

批准号：
17613002
负责人：
SAKURABA Haruhiko
金额：
$ 2.43万
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：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

项目摘要

Genes encoding 2-deoxy-D-ribose-5-phosphate aldolase (DERA) homologues from two hyperthermophiles, the archaeon Pyrobaculum aerophilum and the bacterium Thermotoga maritima, were expressed individually Eschelichia coli, after which the structures and activities of the enzymes produced were characterized and compared with those of E. coli DERA. lb our surprise, the two hyperthermophilic DERAs showed much greater catalysis of sequential aldol condensation using three acetaldehydes as substrates than the E coli enzyme, even at a low temperature (25℃), though both showed much less 2-deoxy-D-ribose-5-phosphate synthetic activity. Both the enzymes were highly resistant to high concentration of acetaldehyde and retained about 50% of their initial activities after 20-hours exposure to 300 mM acetaldehyde at 25℃, whereas the E coli DERA is almost completely inactivated after 2-hours exposure under the same conditions.The structure of the P. aerophilum DERA was determined by x-ray crystallography to a resolution of 2.0A. The mainchain coordinate of the P. aerophilum enzyme monomer was quite similar to those of the T maritime and E call enzymes, whose crystal structures have been already solved. However, the quaternary structure of the hyperthermophilic enzymes was totally different from that of the E coli DERA. The area of the subunit-subunit interface in the dimer of the hyperthermophilic enzymes are much larger compared with the E coif enzyme. This promotes the formation of the unique dimeric structure and strengthens the hydrophobic inter subunit interactions. These structural features are considered to be responsible for the extremely high stability of the hyperthermophilic DERAs.

利用大肠杆菌分别表达了嗜热古菌（Pyrobaculum aerophilum）和海洋热菌（Thermotoga maritima） 2-脱氧-d -核糖-5-磷酸醛dolase （DERA）同源基因，对其结构和活性进行了表征，并与大肠杆菌DERA进行了比较。令我们惊讶的是，即使在较低的温度下（25℃），这两种超嗜热性的DERAs也比大肠杆菌酶表现出更强的以三种乙醛为底物的顺序醛醇缩合的催化作用，尽管它们的2-脱氧-d -核糖-5-磷酸合成活性都要低得多。这两种酶都对高浓度的乙醛具有很强的抗性，在25℃下暴露于300 mM乙醛20小时后仍保持约50%的初始活性，而在相同条件下暴露2小时后，大肠杆菌DERA几乎完全失活。用x射线晶体学测定了嗜气荚膜DERA的结构，分辨率为2.0A。嗜气荚膜酶单体的主链配位与T海事酶和E呼叫酶的主链配位非常相似，这两种酶的晶体结构已被解出。然而，超嗜热酶的四级结构与大肠杆菌DERA完全不同。超嗜热酶二聚体的亚基-亚基界面面积比E - coif酶大得多。这促进了独特二聚体结构的形成，并加强了亚基间的疏水相互作用。这些结构特征被认为是超嗜热性dera具有极高稳定性的原因。