Pavlová Martina, Klvana Martin, Jesenská Andrea, Prokop Zbynek, Konecná Hana, Sato Takashi, Tsuda Masataka, Nagata Yuji, Damborský Jirí
Loschmidt Laboratories, Masaryk University, Brno, Czech Republic.
J Struct Biol. 2007 Feb;157(2):384-92. doi: 10.1016/j.jsb.2006.09.004. Epub 2006 Sep 28.
Haloalkane dehalogenase DhmA from Mycobacterium avium N85 showed poor expression and low stability when produced in Escherichia coli. Here, we present expression DhmA in newly constructed pK4RP rhodococcal expression system in a soluble and stable form. Site-directed mutagenesis was used for the identification of a catalytic pentad, which makes up the reaction machinery of all currently known haloalkane dehalogenases. The putative catalytic triad Asp123, His279, Asp250 and the first halide-stabilizing residue Trp124 were deduced from sequence comparisons. The second stabilizing residue Trp164 was predicted from a homology model. Five point mutants in the catalytic pentad were constructed, tested for activity and were found inactive. A two-step reaction mechanism was proposed for DhmA. Evolution of different types of catalytic pentads and molecular adaptation towards the synthetic substrate 1,2-dichloroethane within the protein family is discussed.
鸟分枝杆菌N85的卤代烷脱卤酶DhmA在大肠杆菌中表达时表现出表达量低和稳定性差的问题。在此，我们展示了DhmA在新构建的pK4RP红球菌表达系统中以可溶且稳定的形式表达。定点诱变用于鉴定催化五元组，其构成了所有目前已知的卤代烷脱卤酶的反应机制。通过序列比较推导了假定的催化三联体Asp123、His279、Asp250以及第一个卤化物稳定残基Trp124。第二个稳定残基Trp164是从同源模型预测得到的。构建了催化五元组中的五个点突变体，测试其活性，发现均无活性。提出了DhmA的两步反应机制。讨论了蛋白质家族中不同类型催化五元组的进化以及对合成底物1,2 - 二氯乙烷的分子适应性。
