Identification of a functionally important loop in Salmonella typhimurium ArnT. Biochemistry 2010 Jan 12;49(1):29-35
Date
12/02/2009Pubmed ID
19947657Pubmed Central ID
PMC2805049DOI
10.1021/bi901572fScopus ID
2-s2.0-73449121971 (requires institutional sign-in at Scopus site) 8 CitationsAbstract
ArnT confers resistance to the antibiotic polymyxin in Salmonella typhimurium and Escherichia coli through 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 from S. 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 alpha-helical and described a new in vivo functional growth assay. Here, we use the cysteine-specific mPEG-mal to demonstrate that all eight of the native cysteines in S. typhimurium ArnT 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 the in vivo growth assay. Following this initial characterization, in vivo expression 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.
Author List
Impellitteri NA, Merten JA, Bretscher LE, Klug CSAuthor
Candice S. Klug PhD Professor in the Biophysics department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Amino Acid SequenceBacterial Proteins
Cell Membrane
Cysteine
Hexosyltransferases
Lipid A
Models, Biological
Molecular Sequence Data
Mutation
Polymyxins
Protein Conformation
Protein Folding
Salmonella typhimurium