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

Abstract

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.

Mechanistic Studies on Dehydration in Class V Lanthipeptides

“…To further investigate the enzyme–substrate interaction, AlphaFold-Multimer was used to generate a predicted structure of the dehydratase complex CaoK–CaoY with the substrate CaoA. − We recently showed for the enzyme TglHI, which performs post-translational modifications on the peptide TglA, that the predicted substrate engagement mechanism was close to that reported crystallographically for a closely related enzyme after the AlphaFold model was made. , The resulting predicted model of CaoK–CaoY had an average predicted local distance difference test (pLDDT) ranging from 84 to 85.6, suggesting high accuracy of the overall prediction (Figure S7A,B and Table S5). In the predicted CaoY structure, the previously discussed lyase active site residues Arg187 and Lys219 are in close proximity, lending support to the predicted structure (Figure S8C).…”

Unusual Dioxygen-Dependent Reactions Catalyzed by Nonheme Iron Enzymes in Natural Product Biosynthesis

“…Cysteine is first loaded on the C-terminus of the TglA peptide via tRNA-dependent amide bond formation catalyzed by TglB. The cysteine moiety of the resulting peptide 247 is next processed into 248 by TglHI (Scheme A). , TglHI is apparently a didomain protein containing the annotated nonheme diiron domain TglH and the leader peptide-binding domain TghI. TghHI reportedly bound 2.5 equiv of Fe, consistent with the annotation of TghHI as a multinuclear iron enzyme.…”

A remarkable transformation catalyzed by a domain-of-unknown-function 692 during the biosynthesis of a new RiPP natural product

“…12,13 One emerging and underexplored group of RiPPs are generated by multinuclear iron-dependent enzymes belonging to the DUF692 family. Members of this enzyme family are structurally related to the triose-phosphate isomerase family, 19 and only two members of the DUF692 enzyme family have been functionally characterized thus far, MbnB 20–24 and TglH. 23,24…”

“…Members of this enzyme family are structurally related to the triose-phosphate isomerase family, 19 and only two members of the DUF692 enzyme family have been functionally characterized thus far, MbnB 20–24 and TglH. 23,24…”