3-Thiaglutamate is a recently identified amino acid analog originating from cysteine. During its biosynthesis, cysteinyl-tRNA is first enzymatically appended to the C-terminus of TglA, a 50-residue ribosomally translated peptide scaffold. After hydrolytic removal of the tRNA, this cysteine residue undergoes modification on the scaffold before eventual proteolysis of the nascent 3-thiaglutamyl residue to release 3-thiaglutamate and regenerate TglA. One of the modifications of TglACys requires a complex of two polypeptides, TglH and TglI, which uses nonheme iron and O 2 to catalyze the removal of the peptidyl-cysteine β-methylene group, oxidation of this Cβ atom to formate, and reattachment of the thiol group to the α carbon. Herein, we use in vitro transcription-coupled translation and expressed protein ligation to characterize the role of the TglA scaffold in TglHI recognition and determine the specificity of TglHI with respect to the C-terminal residues of its substrate TglACys. The results of these experiments establish a synthetically accessible TglACys fragment sufficient for modification by TglHI and identify the l -selenocysteine analog of TglACys, TglASec, as an inhibitor of TglHI. These insights as well as a predicted structure and native mass spectrometry data set the stage for deeper mechanistic investigation of the complex TglHI-catalyzed reaction.
A subset of natural products, such as polyketides and nonribosomal peptides, is biosynthesized while tethered to a carrier peptide via a thioester linkage. Recently, we reported that the biosyntheses of 3-thiaglutamate and ammosamide, single amino acid–derived natural products, employ a very different type of carrier peptide to which the biosynthetic intermediates are bound via an amide linkage. During their biosyntheses, a peptide aminoacyl-transfer ribonucleic acid (tRNA) ligase (PEARL) first loads an amino acid to the C terminus of the carrier peptide for subsequent modification by other enzymes. Proteolytic removal of the modified C-terminal amino acid yields the mature product. We termed natural products that are biosynthesized using such pathways pearlins. To investigate the diversity of pearlins, in this study we experimentally characterized another PEARL-encoding biosynthetic gene cluster (BGC) from Tistrella mobilis ( tmo ). The enzymes encoded in the tmo BGC transformed cysteine into 3-thiahomoleucine both in vitro and in Escherichia coli . During this process, a cobalamin-dependent radical S- adenosylmethionine (SAM) enzyme catalyzes C -isopropylation. This work illustrates that the biosynthesis of amino acid–derived natural products on a carrier peptide is a widespread strategy in nature and expands the spectrum of thiahemiaminal analogs of amino acids that may serve a broader, currently unknown function.
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