Sequential aldol condensation catalyzed by hyperthermophilic 2-deoxy-D-ribose-5-phosphate aldolase
Sequential aldol condensation catalyzed by hyperthermophilic 2-deoxy-D-ribose-5-phosphate aldolase
复制标题
DOI：
10.1128/aem.01101-07
复制
发表时间：
2007-11-01
影响因子：
4.4
通讯作者：
Ohshima, Toshihisa
中科院分区：
文献类型：
--
作者：
Sakuraba, Haruhiko;Yoneda, Kazunari;Ohshima, Toshihisa
从嗜热菌考古杆菌和嗜热细菌中分别表达了编码2-脱氧-D-核糖-5-磷酸缩醛酶(DERA)同系物的基因，并对其结构和活性进行了鉴定，并与E.ColiDERA的结构和活性进行了比较。令我们惊讶的是，即使在较低的温度(25摄氏度)下，这两种超嗜热的DERA对以三种乙醛为底物的顺序羟醛缩合反应的催化作用也比大肠杆菌酶强得多，尽管这两种酶的合成活性都比2-脱氧-D-核糖-5-磷酸低得多。这两种酶对高浓度乙醛都有很高的抗性，在25℃下与300 mM乙醛作用20h后，这两种酶的活性都保持了约50%，而在相同条件下暴露2h后，E.coliDERA几乎完全失活。用X-射线结晶学方法测定了嗜气假单胞菌DERA的结构，分辨率为2.0埃。嗜氧假单胞菌酶单体的主链坐标与毛滴虫和大肠杆菌的主链坐标非常相似，它们的晶体结构已经被解决。然而，高温嗜热酶的四级结构与E.ColiDERA的结构完全不同。嗜热酶二聚体的亚基-亚基界面的面积比大肠杆菌酶的大得多。这促进了独特的二聚体结构的形成，并加强了疏水的亚基间相互作用。这些结构特征被认为是极端嗜热的DERA具有极高稳定性的原因。
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 in Escherichia coli, after which the structures and activities of the enzymes produced were characterized and compared with those of E. coli DERA. To 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 degrees C), although both enzymes showed much less 2-deoxy-D-ribose-5-phosphate synthetic activity. Both the enzymes were highly resistant to high concentrations of acetaldehyde and retained about 50% of their initial activities after a 20-h exposure to 300 mM acetaldehyde at 25 degrees C, whereas the E. coli DERA was almost completely inactivated after a 2-h exposure under the same conditions. The structure of the P. aerophilum DERA was determined by X-ray crystallography to a resolution of 2.0 angstrom. The main chain coordinate of the P. aerophilum enzyme monomer was quite similar to those of the T. maritima and E. coli enzymes, whose crystal structures have already been solved. However, the quaternary structure of the hyperthermophilic enzymes was totally different from that of the E. coli DERA. The areas of the subunit-subunit interface in the dimer of the hyperthermophilic enzymes are much larger than that of the E. coli enzyme. This promotes the formation of the unique dimeric structure and strengthens the hydrophobic intersubunit interactions. These structural features are considered responsible for the extremely high stability of the hyperthermophilic DERAs.