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

分别表达了嗜热菌考古杆菌和嗜热细菌的2-脱氧-D-核糖-5-磷酸缩醛酶(DERA)同源物基因，并对其结构和活性进行了鉴定，并与大肠杆菌DERA进行了比较。令人惊讶的是，即使在较低的温度(25℃)下，这两个极端嗜热的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，尽管两者的合成活性都比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强的耐受性，在25℃下与300 mM乙醛作用20小时后，活性仍保持在原来的50%左右，而在相同条件下暴露2小时后，大肠杆菌DERA几乎完全失活。嗜氧假单胞菌酶单体的主链坐标与T Sea和E Call酶的主链坐标非常相似，它们的晶体结构已经被解决。然而，这些嗜热酶的四级结构与E.ColiDERA完全不同。与E-coif酶相比，高温酶二聚体的亚基-亚基界面的面积要大得多。这促进了独特的二聚体结构的形成，并加强了亚基之间的疏水相互作用。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 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.