Publications

• Structural basis for non-radical catalysis by TsrM, a radical SAM methylase.

  Knox H.L., Chen P.Y., Blaszczyk A.J., Mukherjee A., Grove T.L., Schwalm E.L., Wang B., Drennan C.L., Booker S.J.

  Tryptophan 2C methyltransferase (TsrM) methylates C2 of the indole ring of L-tryptophan during biosynthesis of the quinaldic acid moiety of thiostrepton. TsrM is annotated as a cobalamin-dependent radical S-adenosylmethionine (SAM) methylase; however, TsrM does not reductively cleave SAM to the un ... >> More

  Tryptophan 2C methyltransferase (TsrM) methylates C2 of the indole ring of L-tryptophan during biosynthesis of the quinaldic acid moiety of thiostrepton. TsrM is annotated as a cobalamin-dependent radical S-adenosylmethionine (SAM) methylase; however, TsrM does not reductively cleave SAM to the universal 5'-deoxyadenosyl 5'-radical intermediate, a hallmark of radical SAM (RS) enzymes. Herein, we report structures of TsrM from Kitasatospora setae, which are the first structures of a cobalamin-dependent radical SAM methylase. Unexpectedly, the structures show an essential arginine residue that resides in the proximal coordination sphere of the cobalamin cofactor, and a [4Fe-4S] cluster that is ligated by a glutamyl residue and three cysteines in a canonical CXXXCXXC RS motif. Structures in the presence of substrates suggest a substrate-assisted mechanism of catalysis, wherein the carboxylate group of SAM serves as a general base to deprotonate N1 of the tryptophan substrate, facilitating the formation of a C2 carbanion. << Less

  Nat Chem Biol 17:485-491(2021) [PubMed] [EuropePMC]

• Efficient methylation of C2 in l-tryptophan by the cobalamin-dependent radical <i>S</i>-adenosylmethionine methylase TsrM requires an unmodified N1 amine.

  Blaszczyk A.J., Wang B., Silakov A., Ho J.V., Booker S.J.

  TsrM catalyzes the methylation of C2 in l-tryptophan (Trp). This reaction is the first step in the biosynthesis of the quinaldic acid moiety of the thiopeptide antibiotic thiostrepton, which exhibits potent activity against Gram-positive pathogens. TsrM is a member of the radical <i>S</i>-adenosyl ... >> More

  TsrM catalyzes the methylation of C2 in l-tryptophan (Trp). This reaction is the first step in the biosynthesis of the quinaldic acid moiety of the thiopeptide antibiotic thiostrepton, which exhibits potent activity against Gram-positive pathogens. TsrM is a member of the radical <i>S</i>-adenosylmethionine (SAM) superfamily of enzymes, but it does not catalyze the formation of 5'-deoxyadenosin-5'-yl or any other SAM-derived radical. In addition to a [4Fe-4S] cluster, TsrM contains a cobalamin cofactor that serves as an intermediate methyl carrier in its reaction. However, how this cofactor donates a methyl moiety to the Trp substrate is unknown. Here, we showed that the unmodified N1 position of Trp is important for turnover and that 1-thia-Trp and 1-oxa-Trp serve as competitive inhibitors. We also showed that β-cyclopropyl-Trp undergoes C2 methylation in the absence of cyclopropyl ring opening, disfavoring mechanisms that involve unpaired electron density at C3 of the indole ring. Moreover, we showed that all other indole-substituted analogs of Trp undergo methylation at varying but measurable rates and that the analog 7-aza-Trp, which is expected to temper the nucleophilicity of C2 in Trp, is a very poor substrate. Last, no formation of cob(II)alamin or substrate radicals was observed during the reaction with Trp or any molecule within a tested panel of Trp analogs. In summary, our results are most consistent with a mechanism that involves two polar nucleophilic displacements, the second of which requires deprotonation of the indole nitrogen in Trp during its attack on methylcobalamin. << Less

  J Biol Chem 292:15456-15467(2017) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Formation of 2-methyltryptophan in the biosynthesis of thiostrepton: isolation of S-adenosylmethionine:tryptophan 2-methyltransferase.

  Frenzel T., Zhou P., Floss H.G.

  L-2-Methyltryptophan was found to be an intermediate in the biosynthesis of the antibiotic thiostrepton. It was isolated from growing cultures and resting cells of Streptomyces laurentii in trapping experiments after the application of labeled L-methionine or L-tryptophan. Its formation from L-try ... >> More

  L-2-Methyltryptophan was found to be an intermediate in the biosynthesis of the antibiotic thiostrepton. It was isolated from growing cultures and resting cells of Streptomyces laurentii in trapping experiments after the application of labeled L-methionine or L-tryptophan. Its formation from L-tryptophan and S-adenosylmethionine was studied in a cell-free extract of S. laurentii. Although several attempts to purify the soluble methyltransferase by standard methods failed, some of its characteristics could be determined in the crude extract. The enzyme has a sharp pH optimum at pH 7.8. The apparent Km value for S-adenosylmethionine is 120 microM and the Ki value for S-adenosylhomocysteine is 480 microM. The enzyme is not stereoselective with respect to D- or L-tryptophan, but the D-isomer is converted at a slower rate than the L-isomer. Indolepyruvic acid is also methylated, while indole is not a substrate. The methyl group is transferred with retention of its configuration, contrary to most other methyltransferase reactions. << Less

  Arch Biochem Biophys 278:35-40(1990) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Thiostrepton tryptophan methyltransferase expands the chemistry of radical SAM enzymes.

  Pierre S., Guillot A., Benjdia A., Sandstroem C., Langella P., Berteau O.

  Methylation is among the most widespread chemical modifications encountered in biomolecules and has a pivotal role in many major biological processes. In the biosynthetic pathway of the antibiotic thiostrepton A, we identified what is to our knowledge the first tryptophan methyltransferase. We sho ... >> More

  Methylation is among the most widespread chemical modifications encountered in biomolecules and has a pivotal role in many major biological processes. In the biosynthetic pathway of the antibiotic thiostrepton A, we identified what is to our knowledge the first tryptophan methyltransferase. We show that it uses unprecedented chemistry to methylate inactivated sp(2)-hybridized carbon atoms, despite being predicted to be a radical SAM enzyme. << Less

  Nat. Chem. Biol. 8:957-959(2012) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Spectroscopic and Electrochemical Characterization of the Iron-Sulfur and Cobalamin Cofactors of TsrM, an Unusual Radical S-Adenosylmethionine Methylase.

  Blaszczyk A.J., Silakov A., Zhang B., Maiocco S.J., Lanz N.D., Kelly W.L., Elliott S.J., Krebs C., Booker S.J.

  TsrM, an annotated radical S-adenosylmethionine (SAM) enzyme, catalyzes the methylation of carbon 2 of the indole ring of L-tryptophan. Its reaction is the first step in the biosynthesis of the unique quinaldic acid moiety of thiostrepton A, a thiopeptide antibiotic. The appended methyl group deri ... >> More

  TsrM, an annotated radical S-adenosylmethionine (SAM) enzyme, catalyzes the methylation of carbon 2 of the indole ring of L-tryptophan. Its reaction is the first step in the biosynthesis of the unique quinaldic acid moiety of thiostrepton A, a thiopeptide antibiotic. The appended methyl group derives from SAM; however, the enzyme also requires cobalamin and iron-sulfur cluster cofactors for turnover. In this work we report the overproduction and purification of TsrM and the characterization of its metallocofactors by UV-visible, electron paramagnetic resonance, hyperfine sublevel correlation (HYSCORE), and Mössbauer spectroscopies as well as protein-film electrochemistry (PFE). The enzyme contains 1 equiv of its cobalamin cofactor in its as-isolated state and can be reconstituted with iron and sulfide to contain one [4Fe-4S] cluster with a site-differentiated Fe(2+)/Fe(3+) pair. Our spectroscopic studies suggest that TsrM binds cobalamin in an uncharacteristic five-coordinate base-off/His-off conformation, whereby the dimethylbenzimidazole group is replaced by a non-nitrogenous ligand, which is likely a water molecule. Electrochemical analysis of the protein by PFE indicates a one-electron redox feature with a midpoint potential of -550 mV, which is assigned to a [4Fe-4S](2+)/[4Fe-4S](+) redox couple. Analysis of TsrM by Mössbauer and HYSCORE spectroscopies suggests that SAM does not bind to the unique iron site of the cluster in the same manner as in other radical SAM (RS) enzymes, yet its binding still perturbs the electronic configuration of both the Fe/S cluster and the cob(II)alamin cofactors. These biophysical studies suggest that TsrM is an atypical RS enzyme, consistent with its reported inability to catalyze formation of a 5'-deoxyadenosyl 5'-radical. << Less

  J Am Chem Soc 138:3416-3426(2016) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.