Stanford Advisors

All Publications

  • Substrate Recognition by the Peptidyl-(S)-2-mercaptoglycine Synthase TglHI during 3-Thiaglutamate Biosynthesis. ACS chemical biology McLaughlin, M. I., Yu, Y., van der Donk, W. A. 2022


    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 O2 to catalyze the removal of the peptidyl-cysteine beta-methylene group, oxidation of this Cbeta atom to formate, and reattachment of the thiol group to the alpha 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.

    View details for DOI 10.1021/acschembio.2c00087

    View details for PubMedID 35362960

  • Overall Retention of Methyl Stereochemistry during B-12-Dependent Radical SAM Methyl Transfer in Fosfomycin Biosynthesis BIOCHEMISTRY McLaughlin, M., Pallitsch, K., Wallner, G., van der Donk, W. A., Hammerschmidt, F. 2021; 60 (20): 1587-1596


    Methylcobalamin-dependent radical S-adenosylmethionine (SAM) enzymes methylate non-nucleophilic atoms in a range of substrates. The mechanism of the methyl transfer from cobalt to the receiving atom is still mostly unresolved. Here we determine the stereochemical course of this process at the methyl group during the biosynthesis of the clinically used antibiotic fosfomycin. In vitro reaction of the methyltransferase Fom3 using SAM labeled with 1H, 2H, and 3H in a stereochemically defined manner, followed by chemoenzymatic conversion of the Fom3 product to acetate and subsequent stereochemical analysis, shows that the overall reaction occurs with retention of configuration. This outcome is consistent with a double-inversion process, first in the SN2 reaction of cob(I)alamin with SAM to form methylcobalamin and again in a radical transfer of the methyl group from methylcobalamin to the substrate. The methods developed during this study allow high-yield in situ generation of labeled SAM and recombinant expression and purification of the malate synthase needed for chiral methyl analysis. These methods facilitate the broader use of in vitro chiral methyl analysis techniques to investigate the mechanisms of other novel enzymes.

    View details for DOI 10.1021/acs.biochem.1c00113

    View details for Web of Science ID 000656969900004

    View details for PubMedID 33942609

    View details for PubMedCentralID PMC8158854