Singh K, Kaushik N, Jin J, Madhusudanan M, Modak M J
Department of Biochemistry and Molecular Biology, UMD-New Jersey Medical School and Graduate School of Biomedical Sciences, Newark, NJ 07103, USA.
Protein Eng. 2000 Sep;13(9):635-43. doi: 10.1093/protein/13.9.635.
Gln190 of MuLV reverse transcriptase (RT) plays an important role in the catalytic mechanism of MuLV RT for its conservative and non-conservative mutant derivatives exhibit low catalytic activity. We now report that both Q190N and Q190A MuLV RTs are more efficient in their activity to incorporate ddNTPs and exhibit higher fidelity than the wild-type (WT) enzyme of DNA synthesis in both RNA- and DNA-directed reactions. To obtain some insight into the structural basis for the differential utilization of dNTP and ddNTP by the mutant enzymes, we modeled the binary and the ternary complexes of MuLV RT using corresponding HIV-1 RT structures and available structure of the fragment of MuLV RT. Q190 of MuLV RT appears to be essential for the interaction with 3'OH of dNTP. The lack of a 3'OH moiety in ddNTP does not permit the binding of ddNTPs to WT MuLV RT. However, the shorter side chain of Q190N (or A) mutant MuLV RT and the absence of 3'OH in ddNTP result in the rearrangement of hydrophobic interactions favoring the binding and limited incorporation of ddNTPs. In addition, while modeling the binary and ternary complexes of MuLV RT, we noted that in the formation of the ternary complex, an interaction of Q190 with dNTP substrate requires a shift from its interaction with the template base. This may be achieved by a small conformational change or motion of the loop between beta9 and alphaH containing Q190, which may correspond to the conformational change step requiring participation of Q190 during the catalytic reaction as reported in an earlier biochemical investigation.
莫洛尼氏鼠白血病病毒逆转录酶（RT）的Gln190在莫洛尼氏鼠白血病病毒RT的催化机制中起着重要作用，因为其保守和非保守突变衍生物的催化活性较低。我们现在报告，Q190N和Q190A莫洛尼氏鼠白血病病毒RT在掺入双脱氧核苷酸三磷酸（ddNTPs）的活性方面比野生型（WT）酶更有效，并且在RNA和DNA指导的反应中DNA合成的保真度更高。为了深入了解突变酶对脱氧核苷酸三磷酸（dNTP）和ddNTP差异利用的结构基础，我们使用相应的HIV-1 RT结构和莫洛尼氏鼠白血病病毒RT片段的可用结构对莫洛尼氏鼠白血病病毒RT的二元和三元复合物进行了建模。莫洛尼氏鼠白血病病毒RT的Q190似乎对于与dNTP的3'羟基相互作用至关重要。ddNTP中缺乏3'羟基部分不允许ddNTP与野生型莫洛尼氏鼠白血病病毒RT结合。然而，Q190N（或A）突变莫洛尼氏鼠白血病病毒RT较短的侧链以及ddNTP中不存在3'羟基导致疏水相互作用重新排列，有利于ddNTP的结合和有限掺入。此外，在对莫洛尼氏鼠白血病病毒RT的二元和三元复合物进行建模时，我们注意到在三元复合物形成过程中，Q190与dNTP底物的相互作用需要从其与模板碱基的相互作用转变。这可能通过包含Q190的β9和αH之间环的小构象变化或运动来实现，这可能对应于早期生化研究中报道的催化反应过程中需要Q190参与的构象变化步骤。
