Chen Qihan, Xiao Xiao, Wolfe Aaron, Chen Xiaojiang S
Molecular and Computational Biology Program, Departments of Biological Sciences and Chemistry, University of Southern California, Los Angeles, CA 90089, USA.
Genetic, Molecular and Cellular Biology, Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA.
J Mol Biol. 2016 Jul 3;428(13):2661-70. doi: 10.1016/j.jmb.2016.03.031. Epub 2016 Apr 8.
APOBEC3F (A3F) is a member of the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) family of proteins that can deaminate cytosine (C) to uracil (U) on nucleic acids. A3F is one of the four APOBEC members with two Zn-coordinated homologous cytosine deaminase (CD) domains, with the others being A3G, A3D, and A3B. Here we report the in vitro characterization of DNA binding and deaminase activities using purified wild-type and various mutant proteins of A3F from an Escherichia coli expression system. We show that even though CD1 is catalytically inactive and CD2 is the active deaminase domain, presence of CD1 on the N-terminus of CD2 enhances the deaminase activity by over an order of magnitude. This enhancement of CD2 catalytic activity is mainly through the increase of substrate single-stranded (ss) DNA binding by the N-terminal CD1 domain. We further show that the loop 7 of both CD1 and CD2 of A3F plays an important role for ssDNA binding for each individual domain, as well as for the deaminase activity of CD2 domain in the full-length A3F.
载脂蛋白B mRNA编辑酶催化多肽样（APOBEC）蛋白家族中的载脂蛋白B mRNA编辑酶催化多肽样3F（APOBEC3F，A3F）能够将核酸上的胞嘧啶（C）脱氨基为尿嘧啶（U）。A3F是具有两个锌配位同源胞嘧啶脱氨酶（CD）结构域的四个APOBEC成员之一，其他成员为A3G、A3D和A3B。在此，我们报道了使用来自大肠杆菌表达系统的纯化野生型及各种突变型A3F蛋白对DNA结合活性和脱氨酶活性进行的体外特性分析。我们发现，尽管CD1无催化活性且CD2是活性脱氨酶结构域，但CD1位于CD2的N端可使脱氨酶活性提高一个数量级以上。CD2催化活性的这种增强主要是通过N端CD1结构域增加底物单链（ss）DNA结合实现的。我们进一步表明，A3F的CD1和CD2的环7对于每个结构域的ssDNA结合以及全长A3F中CD2结构域的脱氨酶活性都起着重要作用。
