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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.

分别在大肠杆菌中表达了嗜热古菌Pyrobaculum aerophilum和海洋热袍菌Thermotoga maritima的2-脱氧-D-核糖-5-磷酸醛缩酶（DERA）同源基因，并对所产生的酶的结构和活性进行了表征，并与大肠杆菌的DERA同源基因进行了比较。coli DERA。令人惊讶的是，即使在低温（25℃）下，两种极端嗜热DERA对以三种乙醛为底物的连续羟醛缩合反应的催化作用也比大肠杆菌酶强得多，尽管它们的2-脱氧-D-核糖-5-磷酸合成活性都要低得多。这两种酶对高浓度乙醛有很强的耐受性，在25℃下暴露于300 mM乙醛20 h后，酶活力仍保持在初始活力的50%左右，而E. coliDERA在相同条件下暴露2 h后几乎完全失活。嗜气假单胞菌酶单体的主链配位与海洋嗜热菌和E call酶的主链配位非常相似，后者的晶体结构已经解析。然而，超嗜热酶的四级结构与大肠杆菌DERA的四级结构完全不同。与大肠杆菌酶相比，超嗜热酶二聚体中亚基-亚基界面的面积大得多。这促进了独特的二聚体结构的形成，并加强了亚基间的疏水相互作用。这些结构特征被认为是负责极高的稳定性的超嗜热DERA。