title: N-terminal protein modification using simple aminoacyl transferase substrates.
abstract: Methods for synthetically manipulating protein structure enable greater flexibility in the study of protein function. Previous characterization of the Escherichia coli aminoacyl tRNA transferase (AaT) has shown that it can modify the N-terminus of a protein with an amino acid from a tRNA or a synthetic oligonucleotide donor. Here, we demonstrate that AaT can efficiently use a minimal adenosine substrate, which can be synthesized in one to two steps from readily available starting materials. We have characterized the enzymatic activity of AaT with aminoacyl adenosyl donors and found that reaction products do not inhibit AaT. The use of adenosyl donors removes the substrate limitations imposed by the use of synthetases for tRNA charging and avoids the complex synthesis of an oligonucleotide donor. Thus, our AaT donors increase the potential substrate scope and reaction scale for N-terminal protein modification under conditions that maintain folding.
authors: Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323, United States.
authors: Anne M
authors: AM
authors: Wagner
authors: Mark W
authors: MW
authors: Fegley
authors: John B
authors: JB
authors: Warner
authors: Christina L J
authors: CL
authors: Grindley
authors: Nicholas P
authors: NP
authors: Marotta
authors: E James
authors: EJ
authors: Petersson
mesh_terms: D000241:Adenosine / Q000737:chemistry / Q000378:metabolism*; D019881:Aminoacyltransferases / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D002364:Caseins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D015394:Molecular Structure; D013379:Substrate Specificity
chemical_list: D002364:Caseins; D019881:Aminoacyltransferases; C019675:tRNA-protein transferase, amino acyl; D000241:Adenosine