Adenylation domains CcbC and LmbC control the specific incorporation of amino acid precursors in the biosynthesis of lincosamide antibiotics celesticetin and lincomycin. Both proteins originate from a common L-proline-specific ancestor, but LmbC was evolutionary adapted to use an unusual substrate, (2S,4R)-4-propyl-proline (PPL). Using site-directed mutagenesis of the LmbC substrate binding pocket and an ATP-[32P]PPi exchange assay, three residues, G308, A207 and L246, were identified as crucial for the PPL activation, presumably forming together a channel of a proper size, shape and hydrophobicity to accommodate the propyl side chain of PPL. Subsequently, we experimentally simulated the molecular evolution leading from L-proline-specific substrate binding pocket to the PPL-specific LmbC. The mere change of three amino acid residues in originally strictly L-proline-specific CcbC switched its substrate specificity to prefer PPL and even synthetic alkyl-L-proline derivatives with prolonged side chain. This is the first time that such a comparative study provided an evidence of the evolutionary relevant adaptation of the adenylation domain substrate binding pocket to a new sterically different substrate by a few point mutations. The herein experimentally simulated rearrangement of the substrate binding pocket seems to be the general principle of the de novo genesis of adenylation domains' unusual substrate specificities. However, to keep the overall natural catalytic efficiency of the enzyme, a more comprehensive rearrangement of the whole protein would probably be employed within natural evolution process.

Institute of Microbiology of the Czech Academy of Sciences Prague Czech Republic

Zobrazit více v PubMed

Zhao Q, Wang M, Xu D, Zhang Q, Liu W. Metabolic coupling of two small-molecule thiols programs the biosynthesis of lincomycin A. Nature. Nature Publishing Group; 2015;518: 115–119. doi: 10.1038/nature14137 PubMed DOI

Jiraskova P, Gazak R, Kamenik Z, Steiningerova L, Najmanova L, Kadlcik S, et al. New Concept of the Biosynthesis of 4-Alkyl-L-Proline Precursors of Lincomycin, Hormaomycin, and Pyrrolobenzodiazepines: Could a γ-Glutamyltransferase Cleave the C–C Bond? Front Microbiol. 2016;7: 1–14. doi: 10.3389/fmicb.2016.00001 PubMed DOI PMC

Kadlcik S, Kucera T, Chalupska D, Gazak R, Koberska M, Ulanova D, et al. Adaptation of an L-proline adenylation domain to use 4-propyl-L-proline in the evolution of lincosamide biosynthesis. PLoS One. 2013;8: 1–16. doi: 10.1371/journal.pone.0084902 PubMed DOI PMC

Magerlein BJ. Modification of lincomycin. Adv Appl Microbiol. 1971;14: 185–229. doi: 10.1016/S0065-2164(08)70544-6 PubMed DOI

Kadlcik S, Kamenik Z, Vasek D, Nedved M, Janata J. Elucidation of salicylate attachment in celesticetin biosynthesis opens the door to create a library of more efficient hybrid lincosamide antibiotics. Chem Sci. 2017;8: 3349–3355. doi: 10.1039/c6sc04235j PubMed DOI PMC

Ulanova D, Novotna J, Smutna Y, Kamenik Z, Gazak R, Sulc M, et al. Mutasynthesis of lincomycin derivatives with activity against drug-resistant staphylococci. Antimicrob Agents Chemother. 2010;54: 927–930. doi: 10.1128/AAC.00918-09 PubMed DOI PMC

Janata J, Kadlcik S, Koberska M, Ulanova D, Kamenik Z, Novak P, et al. Lincosamide synthetase—a unique condensation system combining elements of nonribosomal peptide synthetase and mycothiol metabolism. PLoS One. 2015;10: 1–27. doi: 10.1371/journal.pone.0118850 PubMed DOI PMC

Koberska M, Kopecky J, Olsovska J, Jelinkova M, Ulanova D, Man P, et al. Sequence analysis and heterologous expression of the lincomycin biosynthetic cluster of the type strain Streptomyces lincolnensis ATCC 25466. Folia Microbiol (Praha). 2008;53: 395–401. doi: 10.1007/s12223-008-0060-8 PubMed DOI

Conti E, Stachelhaus T, Marahiel M, Brick P. Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S. Embo J. 1997;16: 4174–4183. doi: 10.1093/emboj/16.14.4174 PubMed DOI PMC

Stachelhaus T, Mootz D, Marahiel A. The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases. Chem Biol. 1999;6: 493–505. doi: 10.1016/S1074-5521(99)80082-9 PubMed DOI

Challis GL, Ravel J, Townsend C. Predictive, structure-based model of amino acid recognition by nonribosomal peptide synthetase adenylation domains. Chem Biol. 2000;7: 211–224. doi: 10.1016/S1074-5521(00)00091-0 PubMed DOI

Kyselkova M, Janata J, Sagova-Mareckova M, Kopecky J. Subunit-subunit interactions are weakened in mutant forms of acetohydroxy acid synthase insensitive to valine inhibition. Arch Microbiol. 2010;192: 195–200. doi: 10.1007/s00203-010-0545-0 PubMed DOI

Méjean A, Mann S, Vassiliadis G, Lombard B, Loew D, Ploux O. In vitro reconstitution of the first steps of anatoxin-a biosynthesis in Oscillatoria PCC 6506: From free L-proline to acyl carrier protein bound dehydroproline. Biochemistry. 2010;49: 103–113. doi: 10.1021/bi9018785 PubMed DOI

Kopp M, Irschik H, Gemperlein K, Buntin K, Meiser P, Weissman KJ, et al. Insights into the complex biosynthesis of the leupyrrins in Sorangium cellulosum So ce690. Mol Biosyst. 2011;7: 1549–1563. doi: 10.1039/c0mb00240b PubMed DOI

Garneau S, Dorrestein PC, Kelleher NL, Walsh CT. Characterization of the formation of the pyrrole moiety during clorobiocin and coumermycin A1 biosynthesis. Biochemistry. 2005;44: 2770–2780. doi: 10.1021/bi0476329 PubMed DOI

Zhang X, Parry RJ. Cloning and characterization of the pyrrolomycin biosynthetic gene clusters from Actinosporangium vitaminophilum ATCC 31673 and Streptomyces sp. strain UC 11065. Antimicrob Agents Chemother. 2007;51: 946–957. doi: 10.1128/AAC.01214-06 PubMed DOI PMC

Harris AKP, Williamson NR, Slater H, Cox A, Abbasi S, Foulds I, et al. The Serratia gene cluster encoding biosynthesis of the red antibiotic, prodigiosin, shows species- and strain-dependent genome context variation. Microbiology. 2004;150: 3547–3560. doi: 10.1099/mic.0.27222-0 PubMed DOI

Eppelmann K, Stachelhaus T, Marahiel M. Exploitation of the selectivity-conferring code of nonribosomal peptide synthetases for the rational design of novel peptide antibiotics. Biochemistry. 2002;41: 9718–9726. doi: 10.1021/bi0259406 PubMed DOI

Cheng-Yu C, Ivelin G, Anderson AC, Donald BR. Computational structure-based redesign of enzyme activity. Proc Natl Acad Sci. 2009;106: 3764–3769. doi: 10.1073/pnas.0900266106 PubMed DOI PMC

Thirlway J, Lewis R, Nunns L, Al Nakeeb M, Styles M, Struck AW, et al. Introduction of a non-natural amino acid into a nonribosomal peptide antibiotic by modification of adenylation domain specificity. Angew Chemie—Int Ed. 2012;51: 7181–7184. doi: 10.1002/anie.201202043 PubMed DOI

Bian X, Plaza A, Yan F, Zhang Y, Müller R. Rational and efficient site-directed mutagenesis of adenylation domain alters relative yields of luminmide derivatives in vivo. Biotechnol Bioeng. 2015;112: 1343–1353. doi: 10.1002/bit.25560 PubMed DOI

Kries H, Wachtel R, Pabst A, Wanner B, Niquille D, Hilvert D. Reprogramming nonribosomal peptide synthetases for “clickable” amino acids. Angew Chemie—Int Ed. 2014;53: 10105–10108. doi: 10.1002/anie.201405281 PubMed DOI

Cieślak J, Miyanaga A, Takaku R, Takaishi M, Amagai K, Kudo F, et al. Biochemical characterization and structural insight into aliphatic β-amino acid adenylation enzymes IdnL1 and CmiS6. Proteins Struct Funct Bioinforma. 2017;85: 1238–1247. doi: 10.1002/prot.25284 PubMed DOI

Sieber SA, Marahiel MA. Molecular mechanisms underlying nonribosomal peptide synthesis: Approaches to new antibiotics. Chem Rev. 2005;105: 715–738. doi: 10.1021/cr0301191 PubMed DOI

Zhang K, Nelson KM, Bhuripanyo K, Grimes KD, Zhao B, Aldrich CC, et al. Engineering the substrate specificity of the dhbe adenylation domain by yeast cell surface display. Chem Biol. Elsevier Ltd; 2013;20: 92–101. doi: 10.1016/j.chembiol.2012.10.020 PubMed DOI PMC

Villiers B, Hollfelder F. Directed evolution of a gatekeeper domain in nonribosomal peptide synthesis. Chem Biol. Elsevier Ltd; 2011;18: 1290–1299. doi: 10.1016/j.chembiol.2011.06.014 PubMed DOI

Ushimaru R, Lin C-I, Sasaki E, Liu H. Characterization of Enzymes Catalyzing Transformations of Cysteine S-Conjugated Intermediates in the Lincosamide Biosynthetic Pathway. ChemBioChem. 2016;17: 1606–1611. doi: 10.1002/cbic.201600223 PubMed DOI PMC

Wang M, Zhao Q, Zhang Q, Liu W. Differences in PLP-Dependent Cysteinyl Processing Lead to Diverse S-Functionalization of Lincosamide Antibiotics. J Am Chem Soc. 2016;138: 6348–6351. doi: 10.1021/jacs.6b01751 PubMed DOI

Kamenik Z, Kadlcik S, Radojevic B, Jiraskova P, Kuzma M, Gazak R, et al. Deacetylation of mycothiol-derived “waste product” triggers the last biosynthetic steps of lincosamide antibiotics. Chem Sci. Royal Society of Chemistry; 2016;7: 430–435. doi: 10.1039/c5sc03327f PubMed DOI PMC

Fewer DP, Rouhiainen L, Jokela J, Wahlsten M, Laakso K, Wang H, et al. Recurrent adenylation domain replacement in the microcystin synthetase gene cluster. BMC Evol Biol. 2007;7: 1–11. doi: 10.1186/1471-2148-7-1 PubMed DOI PMC

Ishida K, Welker M, Christiansen G, Cadel-Six S, Bouchier C, Dittmann E, et al. Plasticity and evolution of aeruginosin biosynthesis in cyanobacteria. Appl Environ Microbiol. 2009;75: 2017–2026. doi: 10.1128/AEM.02258-08 PubMed DOI PMC

Crüsemann M, Kohlhaas C, Piel J. Evolution-guided engineering of nonribosomal peptide synthetase adenylation domains. Chem Sci. 2013;4: 1041–1045. doi: 10.1039/b000000x DOI

Höfer I, Crüsemann M, Radzom M, Geers B, Flachshaar D, Cai X, et al. Insights into the biosynthesis of hormaomycin, an exceptionally complex bacterial signaling metabolite. Chem Biol. 2011;18: 381–391. doi: 10.1016/j.chembiol.2010.12.018 PubMed DOI

Gerratana B. Biosynthesis, synthesis, and biological activities of pyrrolobenzodiazepines. Med Res Rev. 2012;32: 254–293. doi: 10.1002/med.20212 PubMed DOI PMC

Kamenik Z, Kadlcik S, Gazak R, Vobruba S, Palanova L, Kuzma M, et al. Diversity of alkylproline moieties in pyrrolobenzodiazepines arises from postcondensation modifications of a unified building block. ACS Chem Biol. 2017;12: 1993–1998. doi: 10.1021/acschembio.7b00335 PubMed DOI

Hu Y, Phelan V, Ntai I, Farnet CM, Zazopoulos E, Bachmann BO. Benzodiazepine biosynthesis in Streptomyces refuineus. Chem Biol. 2007;14: 691–701. doi: 10.1016/j.chembiol.2007.05.009 PubMed DOI

Li W, Khullar A, Chou S, Sacramo A, Gerratana B. Biosynthesis of sibiromycin, a potent antitumor antibiotic. Appl Environ Microbiol. 2009;75: 2869–2878. doi: 10.1128/AEM.02326-08 PubMed DOI PMC

Li W, Chou S, Khullar A, Gerratana B. Cloning and characterization of the biosynthetic gene cluster for tomaymycin, an SJG-136 monomeric analog. Appl Environ Microbiol. 2009;75: 2958–2963. doi: 10.1128/AEM.02325-08 PubMed DOI PMC

Najmanova L, Ulanova D, Jelinkova M, Kamenik Z, Kettnerova E, Koberska M. Sequence analysis of porothramycin biosynthetic gene cluster. Folia Microbiol (Praha). 2014;59: 543–552. doi: 10.1007/s12223-014-0339-x PubMed DOI PMC