Identification of a functionally important loop in Salmonella typhimurium ArnT.
Identification of a functionally important loop in Salmonella typhimurium ArnT.
复制标题
鼠伤寒沙门氏菌 ArnT 中功能重要环的鉴定。
DOI：
10.1021/bi901572f
复制
发表时间：
2010
期刊：
影响因子：
2.9
通讯作者：
Klug,CandiceS
中科院分区：
文献类型：
--
作者：
Impellitteri,NicholasA;Merten,JacquelineA;Bretscher,LynnE;Klug,CandiceS
ArnT confers resistance to the antibiotic polymyxin inSalmonella typhimuriumandEscherichia colithrough the modification of lipid A, a major component of the outer surface of Gram-negative bacteria. ArnT transfers a neutral aminoarabinose moiety onto the negative phosphate groups of lipid A, reducing the surface charge of the bacteria and preventing cationic peptides such as polymyxin from electrostatically recognizing and killing the bacteria. We previously reported the first expression, purification, and functional analysis of ArnT fromS. typhimurium[Bretscher, L. E., Morrell, M. T., Funk, A. L., and Klug, C. S. (2006)Protein Expression Purif. 46, 33−39]. Our studies showed that ArnT is highly α-helical and described a newin vivofunctional growth assay. Here, we use the cysteine-specific mPEG-mal to demonstrate that all eight of the native cysteines inS. typhimuriumArnT are in the reduced form and not involved in disulfide bonds and show that the cysteine-free protein is structurally and functionally intact as characterized by circular dichroism and thein vivogrowth assay. Following this initial characterization,in vivoexpression and function profiles were surveyed for 31 consecutive mutations within a putative ArnT loop. These studies identify for the first time 14 residues that are essential for function of the ArnT transferase and 3 additional residues that completely disrupt protein folding or insertion into the bacterial inner membrane.
