Abstract
d-amino acids endow peptides with diverse, desirable properties, but the post-translational and site-specific epimerization of l-amino acids into their d-counterparts is rare and chemically challenging. Bottromycins are ribosomally synthesized and post-translationally modified peptides that have overcome this challenge and feature a d-aspartate (d-Asp), which was proposed to arise spontaneously during biosynthesis. We have identified the highly unusual α/β-hydrolase (ABH) fold enzyme BotH as a peptide epimerase responsible for the post-translational epimerization of l-Asp to d-Asp during bottromycin biosynthesis. The biochemical characterization of BotH combined with the structures of BotH and the BotH–substrate complex allowed us to propose a mechanism for this reaction. Bioinformatic analyses of BotH homologs show that similar ABH enzymes are found in diverse biosynthetic gene clusters. This places BotH as the founding member of a group of atypical ABH enzymes that may be able to epimerize non-Asp stereocenters across different families of secondary metabolites.
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Data availability
Atomic coordinates and structure factors have been deposited in the RCSB Protein Data Bank with accession codes 6T6H (BotH apo), 6T6X (BotH–3a complex), 6T6Y (BotH–2 complex), 6T6Z (BotH–5 complex) and 6T70 (BotH–6 complex). Other relevant data supporting the findings of this study are available in this published article, its Supplementary Information files or from the corresponding author upon request.
Code availability
Custom scripts used by the authors are available from the corresponding author upon request.
Change history
15 July 2020
A Correction to this paper has been published: https://doi.org/10.1038/s41589-020-0624-8
References
Ollis, D. L. et al. The alpha/beta hydrolase fold. Protein Eng. 5, 197–211 (1992).
Nardini, M. & Dijkstra, B. W. Alpha/beta hydrolase fold enzymes: the family keeps growing. Curr. Opin. Struct. Biol. 9, 732–737 (1999).
Mindrebo, J. T., Nartey, C. M., Seto, Y., Burkart, M. D. & Noel, J. P. Unveiling the functional diversity of the alpha/beta hydrolase superfamily in the plant kingdom. Curr. Opin. Struct. Biol. 41, 233–246 (2016).
Rauwerdink, A. & Kazlauskas, R. J. How the same core catalytic machinery catalyzes 17 different reactions: the serine-histidine-aspartate catalytic triad of alpha/beta-hydrolase fold enzymes. ACS Catal. 5, 6153–6176 (2015).
Hamiaux, C. et al. DAD2 is an alpha/beta hydrolase likely to be involved in the perception of the plant branching hormone, strigolactone. Curr. Biol. 22, 2032–2036 (2012).
Guo, Y., Zheng, Z., La Clair, J. J., Chory, J. & Noel, J. P. Smoke-derived karrikin perception by the alpha/beta-hydrolase KAI2 from Arabidopsis. Proc. Natl Acad. Sci. USA 110, 8284–8289 (2013).
Shimada, A. et al. Structural basis for gibberellin recognition by its receptor GID1. Nature 456, 520–523 (2008).
Marchot, P. & Chatonnet, A. Enzymatic activity and protein interactions in alpha/beta hydrolase fold proteins: moonlighting versus promiscuity. Protein Pept. Lett. 19, 132–143 (2012).
Crone, W. J. K., Leeper, F. J. & Truman, A. W. Identification and characterisation of the gene cluster for the anti-MRSA antibiotic bottromycin: expanding the biosynthetic diversity of ribosomal peptides. Chem. Sci. 3, 3516–3521 (2012).
Gomez-Escribano, J. P., Song, L., Bibb, M. J. & Challis, G. L. Posttranslational β-methylation and macrolactamidination in the biosynthesis of the bottromycin complex of ribosomal peptide antibiotics. Chem. Sci. 3, 3522–3525 (2012).
Hou, Y. et al. Structure and biosynthesis of the antibiotic bottromycin D. Org. Lett. 14, 5050–5053 (2012).
Huo, L., Rachid, S., Stadler, M., Wenzel, S. C. & Muller, R. Synthetic biotechnology to study and engineer ribosomal bottromycin biosynthesis. Chem. Biol. 19, 1278–1287 (2012).
Waisvisz, J. M., van der Hoeven, M. G., van Peppen, J. & Zwennis, W. C. M. Bottromycin. I. A new sulfur-containing antibiotic. J. Am. Chem. Soc. 79, 4520–4521 (1957).
Nakamura, S., Chikaike, T., Karasawa, K., Tanaka, N., Yonehara, H. & Umezawai, H. Isolation and characterization of bottromycins A and B. J. Antibiot. A 18, 47–52 (1965).
Sowa, S. et al. Susceptibility of methicillin-resistant Staphylococcus aureus clinical isolates to various antimicrobial agents. Hiroshima J. Med. Sci. 40, 137–144 (1991).
Shimamura, H. et al. Structure determination and total synthesis of bottromycin A2: a potent antibiotic against MRSA and VRE. Angew. Chem. Int. Ed. Engl. 48, 914–917 (2009).
Otaka, T. & Kaji, A. Mode of action of bottromycin A2. Release of aminoacyl- or peptidyl-tRNA from ribosomes. J. Biol. Chem. 251, 2299–2306 (1976).
Otaka, T. & Kaji, A. Mode of action of bottromycin A2: effect on peptide bond formation. FEBS Lett. 123, 173–176 (1981).
Otaka, T. & Kaji, A. Mode of action of bottromycin A2: effect of bottromycin A2 on polysomes. FEBS Lett. 153, 53–59 (1983).
Arnison, P. G. et al. Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat. Prod. Rep. 30, 108–160 (2013).
Franz, L., Adam, S., Santos-Aberturas, J., Truman, A. W. & Koehnke, J. Macroamidine formation in bottromycins is catalyzed by a divergent YcaO enzyme. J. Am. Chem. Soc. 139, 18158–18161 (2017).
Schwalen, C. J. et al. In vitro biosynthetic studies of bottromycin expand the enzymatic capabilities of the YcaO superfamily. J. Am. Chem. Soc. 139, 18154–18157 (2017).
Crone, W. J. et al. Dissecting bottromycin biosynthesis using comparative untargeted metabolomics. Angew. Chem. Int. Ed. Engl. 55, 9639–9643 (2016).
Mann, G. et al. Structure and substrate recognition of the bottromycin maturation enzyme BotP. Chembiochem 17, 2286–2292 (2016).
Sikandar, A., Franz, L., Melse, O., Antes, I. & Koehnke, J. Thiazoline-specific amidohydrolase PurAH is the gatekeeper of bottromycin biosynthesis. J. Am. Chem. Soc. 141, 9748–9752 (2019).
Hur, G. H., Vickery, C. R. & Burkart, M. D. Explorations of catalytic domains in non-ribosomal peptide synthetase enzymology. Nat. Prod. Rep. 29, 1074–1098 (2012).
Freeman, M. F. et al. Metagenome mining reveals polytheonamides as posttranslationally modified ribosomal peptides. Science 338, 387–390 (2012).
Morinaka, B. I. et al. Radical S-adenosyl methionine epimerases: regioselective introduction of diverse d-amino acid patterns into peptide natural products. Angew. Chem. Int. Ed. Engl. 53, 8503–8507 (2014).
Parent, A. et al. Mechanistic investigations of PoyD, a radical S-adenosyl-l-methionine enzyme catalyzing iterative and directional epimerizations in polytheonamide A biosynthesis. J. Am. Chem. Soc. 140, 2469–2477 (2018).
Benjdia, A., Guillot, A., Ruffie, P., Leprince, J. & Berteau, O. Post-translational modification of ribosomally synthesized peptides by a radical SAM epimerase in Bacillus subtilis. Nat. Chem. 9, 698–707 (2017).
Cotter, P. D. et al. Posttranslational conversion of l-serines to d-alanines is vital for optimal production and activity of the lantibiotic lacticin 3147. Proc. Natl Acad. Sci. USA 102, 18584–18589 (2005).
Yang, X. & van der Donk, W. A. Post-translational Introduction of d-Alanine into ribosomally synthesized peptides by the dehydroalanine reductase NpnJ. J. Am. Chem. Soc. 137, 12426–12429 (2015).
Holm, L. Benchmarking fold detection by DaliLite v.5. Bioinformatics 35, 5326–5327 (2019).
Bains, J., Kaufman, L., Farnell, B. & Boulanger, M. J. A product analog bound form of 3-oxoadipate-enol-lactonase (PcaD) reveals a multifunctional role for the divergent cap domain. J. Mol. Biol. 406, 649–658 (2011).
Gouda, H. et al. Three-dimensional solution structure of bottromycin A2: a potent antibiotic active against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci. Chem. Pharm. Bull. (Tokyo) 60, 169–171 (2012).
Yamada, T. et al. Synthesis and evaluation of antibacterial activity of bottromycins. J. Org. Chem. 83, 7135–7149 (2018).
Jungheim, L. N. et al. Potent human immunodeficiency virus type 1 protease inhibitors that utilize noncoded d-amino acids as P2/P3 ligands. J. Med. Chem. 39, 96–108 (1996).
Rink, R. et al. To protect peptide pharmaceuticals against peptidases. J. Pharmacol. Toxicol. Methods 61, 210–218 (2010).
Hunter, S. et al. InterPro: the integrative protein signature database. Nucleic Acids Res. 37, D211–D215 (2009).
El-Gebali, S. et al. The Pfam protein families database in 2019. Nucleic Acids Res. 47, D427–D432 (2019).
Stachelhaus, T. & Walsh, C. T. Mutational analysis of the epimerization domain in the initiation module PheATE of gramicidin S synthetase. Biochemistry 39, 5775–5787 (2000).
Winter, G. xia2: an expert system for macromolecular crystallography data reduction. J. Appl. Crystallogr. 43, 186–190 (2010).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).
Kabsch, W. Xds. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).
Evans, P. R. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr. D Biol. Crystallogr. 67, 282–292 (2011).
Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr. D Biol. Crystallogr. 69, 1204–1214 (2013).
French, S. & Wilson, K. On the treatment of negative intensity observations. Acta Crystallogr. A Found. Adv. 34, 517–525 (1978).
Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).
Laskowski, R. A. & Swindells, M. B. LigPlot+: multiple ligand–protein interaction diagrams for drug discovery. J. Chem. Inf. Model. 51, 2778–2786 (2011).
Horbal, L., Marques, F., Nadmid, S., Mendes, M. V. & Luzhetskyy, A. Secondary metabolites overproduction through transcriptional gene cluster refactoring. Metab. Eng. 49, 299–315 (2018).
Liu, H. & Naismith, J. H. A simple and efficient expression and purification system using two newly constructed vectors. Protein Expr. Purif. 63, 102–111 (2009).
Mitchell, A. L. et al. InterPro in 2019: improving coverage, classification and access to protein sequence annotations. Nucleic Acids Res. 47, D351–D360 (2019).
Eddy, S. R. Profile hidden Markov models. Bioinformatics 14, 755–763 (1998).
UniProt, C. UniProt: a worldwide hub of protein knowledge. Nucleic Acids Res. 47, D506–D515 (2019).
Blin, K. et al. antiSMASH 4.0—improvements in chemistry prediction and gene cluster boundary identification. Nucleic Acids Res. 45, W36–W41 (2017).
Charif, D. & Lobry, J. R. in Structural Approaches to Sequence Evolution: Molecules, Networks, Populations (eds Bastolla, U. et al.) 207–232 (Springer Berlin Heidelberg, 2007).
Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498–2504 (2003).
Acknowledgements
We thank the Swiss Light Source (X06DA), the European Synchrotron Radiation Facility (ID23–1 and ID23–2) and Deutsches Elektronen Synchrotron (P11), as well as associated beamline staff, for their support. We thank S. Hirono and H. Gouda for providing the coordinate file of their solution NMR structure of bottromycin A2. J.K. is the recipient of an Emmy Noether Fellowship from the Deutsche Forschungsgemeinschaft (grant no. KO 4116/3–2). We thank K. Niefind and R. Guimaraes da Silva for critical reading of the manuscript and helpful suggestions.
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Contributions
A.S. and J.K. established the production and purification of BotH and performed MST experiments. A.S. designed and performed crystallization experiments, determined the reported crystal structures, produced and purified bottromycin A2 and performed pull-down experiments. L.F. established BotH activity, carried out the biochemical experiments, produced BotH substrates, carried out Marfey’s analysis and performed the mass spectrometry. S.A. established the purification of the BotH substrate. J.S.-A. and A.W.T. aided bioinformatic analyses. L.H. produced, purified and analyzed bottromycin A2 and derivatives under the guidance of A.L. O.V.K. designed and performed the bioinformatic analyses and wrote the bioinformatics section. J.K. analyzed and visualized the crystal structures for publication and wrote the paper with contributions from all authors. The full program was carried out under the guidance and direction of J.K.
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Extended data
Extended Data Fig. 1 Crystal structure of BotH.
a, Cartoon representation of apo-BotH. b, Cartoon representation of apo-BotH highlighting the V-shaped loop (magenta) positioned above the active site. c, Electrostatic surface potential of BotH. The Phe-Phe motif at the active-site is shown as blue sticks. d, Superposition of apo-BotH (yellow/magenta) with its closest structural homolog (PDB ID 2xua, cyan) gives a Cα-rmsd of 2.8 Å over 288 residues.
Extended Data Fig. 2 Biosynthesis of compound 3.
Production of 3 can be accomplished in a two-stage, one-pot reaction with quantitative yields.
Extended Data Fig. 3 Spontaneous (non-enzymatic) epimerization of the 3a/b Asp Cα.
A roughly racemic mixture of 3a/b was dissolved in D2O to analyze epimerization. Samples were taken after 2 h, 1 day, 4 days and 6 days. In the acidic (0.1% FA) LC conditions the thaizoline partly (about 50%) re-opens, which leads to the addition of H2O ( + 18.015 Da). Shown mass spectra are averaged from 2.96–3.10 min and 3.10–3.24 min respectively, as the retention times of 3a/b and the respective compound with re-opened thiazoline slightly differ. Shown is the base peak chromatogram (BPC) in H2O. No changes in the shape of the BPCs were observed at the different time points. Representative experiments were repeated independently three times with similar results. BPC = Base Peak Chromatogram.
Extended Data Fig. 4 Crystal structure of BotH in complex with 3.
a, Superposition of the BotH apo structure (green) with the BotH-3a complex structure (cyan). 3a is shown as cyan sticks. b, LigPlus diagram of the interactions between 3a and BotH. 3a is shown with bonds in grey, BotH with bonds in cyan. Water molecules are depicted as cyan spheres, intermolecular hydrogen bonds shown as dashed lines with distances in Å and hydrophobic contacts are shown as red spoked arcs. c, 3a as observed in the complex structure shown as sticks. The intramolecular hydrogen bond is shown as a dashed line with the distance given in Å. d, 3a as observed in the complex crystal structure (left) and Polder map of 3a contoured at 3 σ (right).
Extended Data Fig. 5 BotH single-turn over reaction and the role of water in epimerization.
a, Epimerization under single-turnover conditions in D2O. While the ratio of d-Asp increases significantly (right), the ratio of 1H-d-Asp remains unchanged within experimental error. These data indicate that the majority of protons abstracted by the carboxy group of Asp7 during enamine formation are exchanged with bulk solvent before reprotonation of the enamine intermediate, resulting in deuteron incorporation. Experiments were carried out in triplicate, shown are means ± SD (n = 3) and black dots indicate results for individual measurements. Differences in the fraction of d-Asp at the start and end of the experiment were calculated to be extremely significant (p-value = 0.0008) using an unpaired two tailed t-test. b, Four ordered water molecules surround the carboxy-group of Asp7 and may facilitate proton/deuteron exchange with bulk solvent during the epimerization reaction. O – O distances are given in Å.
Extended Data Fig. 6 Epimerization of Asp7 mutants and their stabilization effect on BotH.
a, b, Epimerization of Asp7 mutants. a, Graphs show extracted ion chromatograms (EICs, calculated mass see Supplementary Table 2 ± 5 ppm) of possible BotH substrates with (red) and without (black) addition of BotH in H2O. No change of the retention time nor an additional peak could be observed in the respective EICs after addition of BotH for mutants Asp7Ala and Asp7Asn, while 3a/b (wt) and Asp7Glu could be epimerized by BotH. b, Mass spectra corresponding to A showing the incorporation of a deuteron for wt and Asp7Glu by BotH in D2O buffer but not for Asp7Ala and Asp7Asn. Representative experiments were repeated independently three times with similar results. c, Thermal shift assays of BotH incubated with different concentrations of wt, Asp7Ala and Asp7Asn substrates. As can be seen, all three substrates lead to a shift in melting temperature at comparable concentrations indicating that all three substrates bind to BotH. The double-peak at 5 µM 3a/b concentration may indicate distinct melting temperatures for 3a- and 3b-complexes. Melting temperatures displayed within the graphs are means ± SD (n = 3).
Extended Data Fig. 7 Binding affinity of BotH-bottromycin derivatives and effect of decarboxylation of 3a on epimerization.
a, Chemical structure of the three bottromycin derivatives used in this study. b, MST measurements to determine the affinities of bottromycin A2 (2) and the three derivatives shown in a for BotH. The affinity of BotH for 2 is 232 ± 81 nM. The additional methyl group at the Cβ position of Val3 of (4)1 reduced the affinity to a KD of 472 ± 62 nM, while the Val3Met mutation of 51 reduced the affinity by an order of magnitude to a KD of 3.2 ± 1.9 µM. Oxidation of the methionine sulfur of 5 (6) resulted in a KD of 459 ± 178 nM. A MS2 spectrum for 6 can be found in Synthetic Procedure 4. Each curve represents three independent samples, data points represent the mean and the error bars represent standard deviations. c, Oxidatively decarboxylated 3a, with deuteron incorporated at the Asp Cα ([M + H]+calc.mono.: 754.3815 Da), was incubated in H2O buffer with (red) and without (black) BotH. BotH is unable to epimerize the decarboxylated 3a as no change of the isotope pattern nor the retention time is observed. This experiments were performed in triplicate with similar results.
Extended Data Fig. 8 Crystal structure of BotH in complex with bottromycin A2 and its derivatives.
a, Bottromycin A2 (2) as observed in the active-site of BotH. Intramolecular hydrogen bonds are shown as dashed lines and distances given in Å. b, Polder maps (grey isomesh) for bottromycin A2 (2) and bottromycin derivatives 5 and 6 bound at the BotH active-site. BotH is shown as a cyan surface representation, the ligands as sticks. The polder maps were contoured at 3 (2), 2.5 (5) and 3 (6) σ. c, Close-up polder map of bottromycin A2 from b shown as a grey isomesh contoured at 3 σ. The structures also provide insights into the varying affinities of the bottromycin derivatives: Based on the BotH-2 complex structure, the additional methyl group of 4 clashes with BotH Glu148, which is involved in hydrogen bonding to the compound and BotH residue Tyr160, which results in a 2-fold weaker affinity. The BotH-5 complex shows that the hydrophobic side-chain of the methionine is forced into a very narrow, highly polar opening lined by Glu148 and His164, which may explain the marked loss in affinity. In the BotH-6 complex structure, the oxidation of the methionine sulfur triggers a rotation of the His164 side-chain, which allows the protein to easily accommodate the bulky ligand side-chain and enables the formation of a new hydrogen bond between the substrate (methionine sulfoxide oxygen) and the side-chain of BotH Arg168. This rationalizes the increase in affinity when compared to unoxidized 5.
Extended Data Fig. 9 Comparison of bottromycin A2 solution structure to bottromycin A2 observed in BotH complex structure.
a, Solution NMR structure of bottromycin A2 (Coordinates available upon request from Prof. Hiroaki Gouda2) shown as yellow sticks. b, Crystal structure of bottromycin A2 bound to BotH shown as grey sticks. The same orientation and magnification is used for A and B. Residues are labeled, Thz8 = Thiazole in position 8. c, Superposition of the solution NMR structure of bottromycin A2 and the bottromycin A2 bound to BotH. Same color scheme as a and b, Pro2 was used as the reference residue. As can be seen, binding to BotH causes a large conformational change and introduces strain. The more relaxed solution state (yellow) clashes with the protein in positions Val3, Phe6 and Thz8. Boxed labels belong to the complex crystal structure, orange arrows indicate movement required to reach unbound state.
Extended Data Fig. 10 Sequence similarity network (SSN) of BotH-like proteins.
SSN for BotH-like proteins lying in or near (closer than 1000 nt) biosynthetic gene clusters (BGCs) is depicted. Nodes represent individual proteins (Accession number given) and colored according to the cluster type (red: RiPP, blue: NRPS, green: PKS, sky: NRPS/PKS, cantaloupe: other), edges represent similarity relationships and are colored according to sequence identity (lighter: less identical, darker: more identical). Edges corresponding to sequence identity below 15% are omitted. BotH is indicated with a star (colored black).
Supplementary information
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Supplementary Figs. 1–8 and Tables 1 and 2, Supplementary Note and references.
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Sikandar, A., Franz, L., Adam, S. et al. The bottromycin epimerase BotH defines a group of atypical α/β-hydrolase-fold enzymes. Nat Chem Biol 16, 1013–1018 (2020). https://doi.org/10.1038/s41589-020-0569-y
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DOI: https://doi.org/10.1038/s41589-020-0569-y
