The catalytic use of a small peptide scaffold for the biosynthesis of amino acid-derived natural products is a recently discovered new biosynthetic strategy. During this process, a peptide-amino acyl tRNA ligase (PEARL) adds amino acids to the C-terminus of a small peptide scaffold in an ATP- and tRNA-dependent process. The mechanism of this unusual transformation is currently not known. In this study, we present a detailed biochemical and mechanistic study of TglB (UniProtKB-F3HQJ1), a PEARL that catalyzes the addition of Cys to the C-terminus of the peptide TglA in the biosynthesis of 3-thiaglutamate in the plant pathogen Pseudomonas syringae. TglB recognizes several important residues close to the C-terminus of TglA to perform its activity and is tolerant with respect to the last amino acid of its substrate peptide. The enzyme recognizes the acceptor stem of tRNACys, as micro- and minihelices, truncated versions of full-length tRNACys that contain the acceptor stem, were also accepted. Mutagenesis of conserved residues in TglB identified several key residues for catalysis and did not support the possibility of TglB adopting various ping-pong mechanisms to catalyze the amino acid addition reaction. Using isotopic labeling studies, we demonstrate that ATP is used to directly phosphorylate the C-terminal carboxylate of TglA. Collectively, the data support a general mechanism for the amino acid addition reaction catalyzed by this class of enzyme.
ASJC Scopus subject areas
- Colloid and Surface Chemistry