Substrate Recognition by the Peptidyl-(S)-2-mercaptoglycineSynthase TglHI during 3-Thiaglutamate Biosynthesis

3-Thiaglutamate is a recently identified amino acidanalog originating from cysteine. During its biosynthesis, cysteinyl-tRNA isfirst enzymatically appended to the C-terminus of TglA, a50-residue ribosomally translated peptide scaffold. After hydrolyticremoval of the tRNA, this cysteine residue undergoes modificationon the scaffold before eventual proteolysis of the nascent 3-thiaglutamyl residue to release 3-thiaglutamate and regenerateTglA. One of the modifications of TglACys requires a complex oftwo polypeptides, TglH and TglI, which uses nonheme iron and O2to catalyze the removal of the peptidyl-cysteine beta-methylene group,oxidation of this C beta atom to formate, and reattachment of the thiolgroup to the alpha carbon. Herein, we usein vitrotranscription-coupled translation and expressed protein ligation to characterizethe role of the TglA scaffold in TglHI recognition and determine the specificity of TglHI with respect to the C-terminal residues ofits substrate TglACys. The results of these experiments establish a synthetically accessible TglACys fragment sufficient formodification by TglHI and identify theL-selenocysteine analog of TglACys, TglASec, as an inhibitor of TglHI. These insights as wellas a predicted structure and native mass spectrometry data set the stage for deeper mechanistic investigation of the complex TglHI-catalyzed reaction.

Automated summary

This study investigates the role of the TglA scaffold in TglHI recognition and determines the specificity of TglHI towards the C-terminal residues of its substrate TglACys using in vitro transcription-coupled translation and expressed protein ligation. The results identify a synthetically accessible TglACys fragment sufficient for modification by TglHI and reveal the L-selenocysteine analog of TglACys, TglASec, as an inhibitor of TglHI.