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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

  • The Fellowship of the Rings: Macrocyclic Antibiotic Peptides Reveal an Anti-Gram-Negative Target. Biochemistry McLaughlin, M. I., van der Donk, W. A. 2020; 59 (4): 343-345

    View details for DOI 10.1021/acs.biochem.9b01086

    View details for PubMedID 31909599

    View details for PubMedCentralID PMC8223768

  • Insights into AMS/PCAT transporters from biochemical and structural characterization of a double Glycine motif protease. eLife Bobeica, S. C., Dong, S. H., Huo, L., Mazo, N., McLaughlin, M. I., Jiménez-Osés, G., Nair, S. K., van der Donk, W. A. 2019; 8


    The secretion of peptides and proteins is essential for survival and ecological adaptation of bacteria. Dual-functional ATP-binding cassette transporters export antimicrobial or quorum signaling peptides in Gram-positive bacteria. Their substrates contain a leader sequence that is excised by an N-terminal peptidase C39 domain at a double Gly motif. We characterized the protease domain (LahT150) of a transporter from a lanthipeptide biosynthetic operon in Lachnospiraceae and demonstrate that this protease can remove the leader peptide from a diverse set of peptides. The 2.0 Å resolution crystal structure of the protease domain in complex with a covalently bound leader peptide demonstrates the basis for substrate recognition across the entire class of such transporters. The structural data also provide a model for understanding the role of leader peptide recognition in the translocation cycle, and the function of degenerate, non-functional C39-like domains (CLD) in substrate recruitment in toxin exporters in Gram-negative bacteria.

    View details for DOI 10.7554/eLife.42305

    View details for PubMedID 30638446

    View details for PubMedCentralID PMC6363468

  • Stereospecific Radical-Mediated B12-Dependent Methyl Transfer by the Fosfomycin Biosynthesis Enzyme Fom3. Biochemistry McLaughlin, M. I., van der Donk, W. A. 2018; 57 (33): 4967-4971


    Fom3, the antepenultimate enzyme in the fosfomycin biosynthetic pathway in Streptomyces spp., is a class B cobalamin-dependent radical SAM methyltransferase that catalyzes methylation of (5'-cytidylyl)-2-hydroxyethylphosphonate (2-HEP-CMP) to form (5'-cytidylyl)-2-hydroxypropylphosphonate (2-HPP-CMP). Previously, the reaction of Fom3 with 2-HEP-CMP produced 2-HPP-CMP with mixed stereochemistry at C2. Mechanistic characterization has been challenging because of insoluble expression and poor cobalamin (B12) incorporation in Escherichia coli. Recently, soluble E. coli expression and incorporation of cobalamin into Fom3 were achieved by overexpression of the BtuCEDFB cobalamin uptake system. Herein, we use this new method to obtain Fom3 from Streptomyces wedmorensis. We show that the initiator 5'-deoxyadenosyl radical stereospecifically abstracts the pro- R hydrogen atom from the C2 position of 2-HEP-CMP and use the downstream enzymes FomD and Fom4 to demonstrate that our preparation of Fom3 produces only (2 S)-2-HPP-CMP. Additionally, we show that the added methyl group originates from SAM under multiple-turnover conditions, but the first turnover uses a methyl donor already present on the enzyme; furthermore, cobalamin isolated from Fom3 reaction mixtures contains methyl groups derived from SAM. These results are consistent with a model in which Fom3 catalyzes methyl transfer from SAM to cobalamin and the resulting methylcobalamin (MeCbl) is the ultimate methyl source for the reaction.

    View details for DOI 10.1021/acs.biochem.8b00616

    View details for PubMedID 29969250

    View details for PubMedCentralID PMC6103835

  • Crystallographic snapshots of sulfur insertion by lipoyl synthase. Proceedings of the National Academy of Sciences of the United States of America McLaughlin, M. I., Lanz, N. D., Goldman, P. J., Lee, K. H., Booker, S. J., Drennan, C. L. 2016; 113 (34): 9446-50


    Lipoyl synthase (LipA) catalyzes the insertion of two sulfur atoms at the unactivated C6 and C8 positions of a protein-bound octanoyl chain to produce the lipoyl cofactor. To activate its substrate for sulfur insertion, LipA uses a [4Fe-4S] cluster and S-adenosylmethionine (AdoMet) radical chemistry; the remainder of the reaction mechanism, especially the source of the sulfur, has been less clear. One controversial proposal involves the removal of sulfur from a second (auxiliary) [4Fe-4S] cluster on the enzyme, resulting in destruction of the cluster during each round of catalysis. Here, we present two high-resolution crystal structures of LipA from Mycobacterium tuberculosis: one in its resting state and one at an intermediate state during turnover. In the resting state, an auxiliary [4Fe-4S] cluster has an unusual serine ligation to one of the irons. After reaction with an octanoyllysine-containing 8-mer peptide substrate and 1 eq AdoMet, conditions that allow for the first sulfur insertion but not the second insertion, the serine ligand dissociates from the cluster, the iron ion is lost, and a sulfur atom that is still part of the cluster becomes covalently attached to C6 of the octanoyl substrate. This intermediate structure provides a clear picture of iron-sulfur cluster destruction in action, supporting the role of the auxiliary cluster as the sulfur source in the LipA reaction and describing a radical strategy for sulfur incorporation into completely unactivated substrates.

    View details for DOI 10.1073/pnas.1602486113

    View details for PubMedID 27506792

    View details for PubMedCentralID PMC5003258

  • Structure of Quinolinate Synthase from Pyrococcus horikoshii in the Presence of Its Product, Quinolinic Acid. Journal of the American Chemical Society Esakova, O. A., Silakov, A., Grove, T. L., Saunders, A. H., McLaughlin, M. I., Yennawar, N. H., Booker, S. J. 2016; 138 (23): 7224-7


    Quinolinic acid (QA) is a common intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD(+)) and its derivatives in all organisms that synthesize the molecule de novo. In most prokaryotes, it is formed from the condensation of dihydroxyacetone phosphate (DHAP) and aspartate-enamine by the action of quinolinate synthase (NadA). NadA contains a [4Fe-4S] cluster cofactor with a unique, non-cysteinyl-ligated, iron ion (Fea), which is proposed to bind the hydroxyl group of a postulated intermediate in the last step of the reaction to facilitate a dehydration. However, direct evidence for this role in catalysis has yet to be provided. Herein, we present the structure of NadA in the presence of the product of its reaction, QA. We find that N1 and the C7 carboxylate group of QA ligate to Fea in a bidentate fashion, which is confirmed by Hyperfine Sublevel Correlation (HYSCORE) spectroscopy. This binding mode would place the C5 hydroxyl group of the postulated final intermediate distal to Fea and virtually incapable of coordinating to it. The structure shows that three strictly conserved amino acids, Glu198, Tyr109, and Tyr23, are in close proximity to the bound product. Substitution of these amino acids with Gln, Phe, and Phe, respectively, leads to complete loss of activity.

    View details for DOI 10.1021/jacs.6b02708

    View details for PubMedID 27224840

  • Allosteric Inhibition of Human Ribonucleotide Reductase by dATP Entails the Stabilization of a Hexamer BIOCHEMISTRY Ando, N., Li, H., Brignole, E. J., Thompson, S., McLaughlin, M. I., Page, J. E., Asturias, F. J., Stubbe, J., Drennan, C. L. 2016; 55 (2): 373-381


    Ribonucleotide reductases (RNRs) are responsible for all de novo biosynthesis of DNA precursors in nature by catalyzing the conversion of ribonucleotides to deoxyribonucleotides. Because of its essential role in cell division, human RNR is a target for a number of anticancer drugs in clinical use. Like other class Ia RNRs, human RNR requires both a radical-generation subunit (β) and nucleotide-binding subunit (α) for activity. Because of their complex dependence on allosteric effectors, however, the active and inactive quaternary forms of many class Ia RNRs have remained in question. Here, we present an X-ray crystal structure of the human α subunit in the presence of inhibiting levels of dATP, depicting a ring-shaped hexamer (α6) where the active sites line the inner hole. Surprisingly, our small-angle X-ray scattering (SAXS) results indicate that human α forms a similar hexamer in the presence of ATP, an activating effector. In both cases, α6 is assembled from dimers (α2) without a previously proposed tetramer intermediate (α4). However, we show with SAXS and electron microscopy that at millimolar ATP, the ATP-induced α6 can further interconvert with higher-order filaments. Differences in the dATP- and ATP-induced α6 were further examined by SAXS in the presence of the β subunit and by activity assays as a function of ATP or dATP. Together, these results suggest that dATP-induced α6 is more stable than the ATP-induced α6 and that stabilization of this ring-shaped configuration provides a mechanism to prevent access of the β subunit to the active site of α.

    View details for DOI 10.1021/acs.biochem.5b01207

    View details for Web of Science ID 000368562100014

    View details for PubMedID 26727048

    View details for PubMedCentralID PMC4722859

  • X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification. Proceedings of the National Academy of Sciences of the United States of America Goldman, P. J., Grove, T. L., Sites, L. A., McLaughlin, M. I., Booker, S. J., Drennan, C. L. 2013; 110 (21): 8519-24


    Arylsulfatases require a maturating enzyme to perform a co- or posttranslational modification to form a catalytically essential formylglycine (FGly) residue. In organisms that live aerobically, molecular oxygen is used enzymatically to oxidize cysteine to FGly. Under anaerobic conditions, S-adenosylmethionine (AdoMet) radical chemistry is used. Here we present the structures of an anaerobic sulfatase maturating enzyme (anSME), both with and without peptidyl-substrates, at 1.6-1.8 Å resolution. We find that anSMEs differ from their aerobic counterparts in using backbone-based hydrogen-bonding patterns to interact with their peptidyl-substrates, leading to decreased sequence specificity. These anSME structures from Clostridium perfringens are also the first of an AdoMet radical enzyme that performs dehydrogenase chemistry. Together with accompanying mutagenesis data, a mechanistic proposal is put forth for how AdoMet radical chemistry is coopted to perform a dehydrogenation reaction. In the oxidation of cysteine or serine to FGly by anSME, we identify D277 and an auxiliary [4Fe-4S] cluster as the likely acceptor of the final proton and electron, respectively. D277 and both auxiliary clusters are housed in a cysteine-rich C-terminal domain, termed SPASM domain, that contains homology to ~1,400 other unique AdoMet radical enzymes proposed to use [4Fe-4S] clusters to ligate peptidyl-substrates for subsequent modification. In contrast to this proposal, we find that neither auxiliary cluster in anSME bind substrate, and both are fully ligated by cysteine residues. Instead, our structural data suggest that the placement of these auxiliary clusters creates a conduit for electrons to travel from the buried substrate to the protein surface.

    View details for DOI 10.1073/pnas.1302417110

    View details for PubMedID 23650368

    View details for PubMedCentralID PMC3666706

  • Structural basis for methyl transfer by a radical SAM enzyme. Science (New York, N.Y.) Boal, A. K., Grove, T. L., McLaughlin, M. I., Yennawar, N. H., Booker, S. J., Rosenzweig, A. C. 2011; 332 (6033): 1089-92


    The radical S-adenosyl-L-methionine (SAM) enzymes RlmN and Cfr methylate 23S ribosomal RNA, modifying the C2 or C8 position of adenosine 2503. The methyl groups are installed by a two-step sequence involving initial methylation of a conserved Cys residue (RlmN Cys(355)) by SAM. Methyl transfer to the substrate requires reductive cleavage of a second equivalent of SAM. Crystal structures of RlmN and RlmN with SAM show that a single molecule of SAM coordinates the [4Fe-4S] cluster. Residue Cys(355) is S-methylated and located proximal to the SAM methyl group, suggesting the SAM that is involved in the initial methyl transfer binds at the same site. Thus, RlmN accomplishes its complex reaction with structural economy, harnessing the two most important reactivities of SAM within a single site.

    View details for DOI 10.1126/science.1205358

    View details for PubMedID 21527678

    View details for PubMedCentralID PMC3506250