title: Semisynthesis and initial characterization of sortase A mutants containing selenocysteine and homocysteine.
abstract: The bacterial transpeptidase sortase A is a well-established tool in protein chemistry and catalyzes the chemoselective ligation of peptides and proteins. During catalysis sortase A cleaves the conserved Leu-Pro-X-Thr-Gly sorting motif at the Thr residue under concomitant thioester formation at active site Cys184. We have used expressed protein ligation (EPL) to generate sortase mutants with Cys184 replaced by selenocysteine (Sec) and homocysteine (Hcy). Sec-sortase showed a moderate 2-3-fold reduction in catalytic activity in contrast to Hcy-sortase which is a poor catalyst with less than 1% of wild-type activity. The sensitivity of the active site nucleophiles towards an alkylation reagent correlated with the pKa values of the mutated residues. Furthermore, the pH-profile of Sec-sortase was shifted to more acidic conditions when compared to the wild-type enzyme. These observations provide information on sortase catalysis and the semisynthetic enzymes might represent useful tools for further biochemical investigations and engineering approaches of sortases A.
authors.affiliation: Interfaculty Institute of Biochemistry, University of Tuebingen, Hoppe-Seyler-Str. 4, D-72076 Tuebingen, Germany.
authors.forename: Lena
authors.initials: L
authors.lastname: Schmohl
authors.affiliation: Interfaculty Institute of Biochemistry, University of Tuebingen, Hoppe-Seyler-Str. 4, D-72076 Tuebingen, Germany.
authors.forename: Felix Roman
authors.initials: FR
authors.lastname: Wagner
authors.affiliation: Laboratory of Mass Spectrometry, Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Strasse 10, D-13125 Berlin, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Schümann
authors.affiliation: Laboratory of Mass Spectrometry, Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Strasse 10, D-13125 Berlin, Germany.
authors.forename: Eberhard
authors.initials: E
authors.lastname: Krause
authors.affiliation: Interfaculty Institute of Biochemistry, University of Tuebingen, Hoppe-Seyler-Str. 4, D-72076 Tuebingen, Germany. Electronic address: dirk.schwarzer@uni-tuebingen.de.
authors.forename: Dirk
authors.initials: D
authors.lastname: Schwarzer
mesh_terms: D000478:Alkylation; D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006710:Homocysteine / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D015202:Protein Engineering; D017510:Protein Folding; D017279:Selenocysteine / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D060327:Solid-Phase Synthesis Techniques; D014170:Transformation, Genetic
chemical_list: D001426:Bacterial Proteins; D017279:Selenocysteine; D006710:Homocysteine; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases
keywords: Expressed protein ligation; Homocysteine; Protein semisynthesis; Selenocysteine; Sortase; Sortase-mediated ligation