Clinically used lincosamide antibiotic lincomycin incorporates in its structure 4-propyl-L-proline (PPL), an unusual amino acid, while celesticetin, a less efficient related compound, makes use of proteinogenic L-proline. Biochemical characterization, as well as phylogenetic analysis and homology modelling combined with the molecular dynamics simulation were employed for complex comparative analysis of the orthologous protein pair LmbC and CcbC from the biosynthesis of lincomycin and celesticetin, respectively. The analysis proved the compared proteins to be the stand-alone adenylation domains strictly preferring their own natural substrate, PPL or L-proline. The LmbC substrate binding pocket is adapted to accommodate a rare PPL precursor. When compared with L-proline specific ones, several large amino acid residues were replaced by smaller ones opening a channel which allowed the alkyl side chain of PPL to be accommodated. One of the most important differences, that of the residue corresponding to V306 in CcbC changing to G308 in LmbC, was investigated in vitro and in silico. Moreover, the substrate binding pocket rearrangement also allowed LmbC to effectively adenylate 4-butyl-L-proline and 4-pentyl-L-proline, substrates with even longer alkyl side chains, producing more potent lincosamides. A shift of LmbC substrate specificity appears to be an integral part of biosynthetic pathway adaptation to the PPL acquisition. A set of genes presumably coding for the PPL biosynthesis is present in the lincomycin--but not in the celesticetin cluster; their homologs are found in biosynthetic clusters of some pyrrolobenzodiazepines (PBD) and hormaomycin. Whereas in the PBD and hormaomycin pathways the arising precursors are condensed to another amino acid moiety, the LmbC protein is the first functionally proved part of a unique condensation enzyme connecting PPL to the specialized amino sugar building unit.

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

Institute of Microbiology Academy of Sciences of the Czech Republic Prague Czech Republic ; Department of Biochemistry and Structural Biology Institute of Molecular Biology Slovac Academy of Sciences Bratislava Slovakia

Zobrazit více v PubMed

Brahme NM, Gonzalez JE, Rolls JP, Hessler EJ, Mizsak S et al. (1984) Biosynthesis of the lincomycins. 1. Studies using stable isotopes on the biosynthesis of the propyl-L-hygric and ethyl-L-hygric acid moieties of lincomycin-A and lincomycin-B. J Am Chem. Soc 106: 7873-7878.

Neusser D, Schmidt H, Spizèk J, Novotnà J, Peschke U et al. (1998) The genes lmbB1 and lmbB2 of Streptomyces lincolnensis encode enzymes involved in the conversion of L-tyrosine to propylproline during the biosynthesis of the antibiotic lincomycin A. Arch Microbiol 169: 322-332. doi:10.1007/s002030050578. PubMed: 9531633. PubMed DOI

Novotná J, Honzátko A, Bednář P, Kopecký J, Janata J et al. (2004) L-3,4-Dihydroxyphenyl alanine-extradiol cleavage is followed by intramolecular cyclization in lincomycin biosynthesis. Eur J Biochem 271: 3678-3683. doi:10.1111/j.1432-1033.2004.04308.x. PubMed: 15355345. PubMed DOI

Hu Y, Phelan V, Ntai I, Farnet CM, Zazopoulos E et al. (2007) Benzodiazepine biosynthesis in Streptomyces refuineus . Chem Biol 14: 691-701. doi:10.1016/j.chembiol.2007.05.009. PubMed: 17584616. PubMed DOI

Li W, Khullar A, Chou S, Sacramo A, Gerratana B (2009) Biosynthesis of sibiromycin, a potent antitumor antibiotic. Appl Environ Microbiol 75: 2869-2878. doi:10.1128/AEM.02326-08. PubMed: 19270142. PubMed DOI PMC

Li W, Chou SC, Khullar A, Gerratana B (2009) Cloning and characterization of the biosynthetic gene cluster for tomaymycin, an SJG-136 monomeric analog. Appl Environ Microbiol 75: 2958-2963. doi:10.1128/AEM.02325-08. PubMed: 19270147. PubMed DOI PMC

Argoudelis AD, Fox JA, Eble TE (1965) U-21,669 - A new lincomycin-related antibiotic. Biochemistry 4: 698-703. doi:10.1021/bi00880a014. PubMed: 14323574. PubMed DOI

Höfer I, Crüsemann M, Radzom M, Geers B, Flachshaar D et al. (2011) Insights into the biosynthesis of hormaomycin, an exceptionally complex bacterial signaling metabolite. Chem. Biol 18: 381-391. PubMed

Chung ST, Manis JJ, McWethy SJ, Patt TE, Witz DF, et al. (1997) Fermentation, biosynthesis, and molecular genetics of lincomycin. In: Strohl WR. Biotechnology of Antibiotics; New York: Drugs and the pharmaceutical sciences. pp. 165–186

Peschke U, Schmidt H, Zhang HZ, Piepersberg W (1995) Molecular characterization of the lincomycin-production gene-cluster of Streptomyces lincolnensis-78-11. Mol Microbiol 16: 1137-1156. doi:10.1111/j.1365-2958.1995.tb02338.x. PubMed: 8577249. PubMed DOI

Koběrská M, Kopecký J, Olšovská J, Jelínková M, Ulanova D et al. (2008) Sequence analysis and heterologous expression of the lincomycin biosynthetic cluster of the type strain Streptomyces lincolnensis ATCC 25466. Folia Microbiol 53: 395-401. doi:10.1007/s12223-008-0060-8. PubMed DOI

Thomas MG, Burkart MD, Walsh CT (2002) Conversion of L-proline to pyrrolyl-2-carboxyl-S-PCP during undecylprodigiosin and pyoluteorin biosynthesis. Chem. Biol 9: 171-184. PubMed

Garneau S, Dorrestein PC, Kelleher NL, Walsh CT (2005) Characterization of the formation of the pyrrole moiety during clorobiocin and coumermycin A1 biosynthesis. Biochemistry 44: 2770-2780. doi:10.1021/bi0476329. PubMed: 15723521. PubMed DOI

Méjean A, Mann S, Vassiliadis G, Lombard B, Loew D et al. (2010) 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 49: 103-113. doi:10.1021/bi9018785. PubMed: 19954230. PubMed DOI

Kopp M, Irschik H, Gemperlein K, Buntin K, Meiser P et al. (2011) Insights into the complex biosynthesis of the leupyrrins in Sorangium cellulosum So ce690. Mol Biosyst 7: 1549-1563. doi:10.1039/c0mb00240b. PubMed: 21365089. PubMed DOI

Marahiel MA, Stachelhaus T, Mootz HD (1997) Modular peptide synthetases involved in nonribosomal peptide synthesis. Chem Rev 97: 2651-2673. doi:10.1021/cr960029e. PubMed: 11851476. PubMed DOI

Wang ZX, Li SM, Heide L (2000) Identification of the coumermycin A1 biosynthetic gene cluster of Streptomyces rishiriensis DSM 40489. Antimicrob Agents Chemother 44: 3040-3048. doi:10.1128/AAC.44.11.3040-3048.2000. PubMed: 11036020. PubMed DOI PMC

Pojer F, Li SM, Heide L (2002) Molecular cloning and sequence analysis of the clorobiocin biosynthetic gene cluster: new insights into the biosynthesis of aminocoumarin antibiotics. Microbiology 148: 3901-3911. PubMed: 12480894. PubMed

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

Müller C, Nolden S, Gebhardt P, Heinzelmann E, Lange C et al. (2007) Sequencing and analysis of the biosynthetic gene cluster of the lipopeptide antibiotic friulimicin in Actinoplanes friuliensis . Antimicrob Agents Chemother 51: 1028-1037. doi:10.1128/AAC.00942-06. PubMed: 17220414. PubMed DOI PMC

Wang Y, Chen Y, Shen QR, Yin XH (2011) Molecular cloning and identification of the laspartomycin biosynthetic gene cluster from Streptomyces viridochromogenes . Gene 483: 11-21. doi:10.1016/j.gene.2011.05.005. PubMed: 21640802. PubMed DOI PMC

de Crécy-Lagard V, Blanc V, Gil P, Naudin L, Lorenzon S et al. (1997) Pristinamycin I biosynthesis in Streptomyces pristinaespiralis: Molecular characterization of the first two structural peptide synthetase genes. J Bacteriol 179: 705-713. PubMed: 9006024. PubMed PMC

Pohle S, Appelt C, Roux M, Fiedler H-P, Süssmuth RD (2011) Biosynthetic gene cluster of the non-ribosomally synthesized cyclodepsipeptide skyllamycin: Deciphering unprecedented ways of unusual hydroxylation reactions. J Am Chem Soc 133: 6194-6205. doi:10.1021/ja108971p. PubMed: 21456593. PubMed DOI

Pfennig F, Schauwecker F, Keller U (1999) Molecular characterization of the genes of actinomycin synthetase I and of a 4-methyl-3-hydroxyanthranilic acid carrier protein involved in the assembly of the acylpeptide chain of actinomycin in Streptomyces . J Biol Chem 274: 12508-12516. doi:10.1074/jbc.274.18.12508. PubMed: 10212227. PubMed DOI

Wenzel SC, Kunze B, Höfle G, Silakowski B, Scharfe M et al. (2005) Structure and biosynthesis of myxochromides S1-3 in Stigmatella aurantiaca: Evidence for an iterative bacterial type I polyketide synthase and for module skipping in nonribosomal peptide biosynthesis. Chembiochem 6: 375-385. doi:10.1002/cbic.200400282. PubMed: 15651040. PubMed DOI

Scholz-Schroeder BK, Soule JD, Gross DC (2003) The sypA, sypB and sypC synthetase genes encode twenty-two modules involved in the nonribosomal peptide synthesis of syringopeptin by Pseudomonas syringae pv. syringae B301D. Mol Plant-Microbe Interact 16: 271-280. doi:10.1094/MPMI.2003.16.4.271. PubMed: 12744455. PubMed DOI

Hoffmann D, Hevel JM, Moore RE, Moore BS (2003) Sequence analysis and biochemical characterization of the nostopeptolide A biosynthetic gene cluster from Nostoc sp GSV224. Gene 311: 171-180. doi:10.1016/S0378-1119(03)00587-0. PubMed: 12853152. PubMed DOI

Luesch H, Hoffmann D, Hevel JM, Becker JE, Golakoti T et al. (2003) Biosynthesis of 4-methylproline in cyanobacteria: Cloning of nosE and nosF genes and biochemical characterization of the encoded dehydrogenase and reductase activities. J Org Chem 68: 83-91. doi:10.1021/jo026479q. PubMed: 12515465. PubMed DOI

Duitman EH, Hamoen LW, Rembold M, Venema G, Seitz H et al. (1999) The mycosubtilin synthetase of Bacillus subtilis ATCC6633: A multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase. Proc Natl Acad Sci U S A 96: 13294-13299. doi:10.1073/pnas.96.23.13294. PubMed: 10557314. PubMed DOI PMC

Tsuge K, Akiyama T, Shoda M (2001) Cloning, sequencing, and characterization of the iturin A operon. J Bacteriol 183: 6265-6273. doi:10.1128/JB.183.21.6265-6273.2001. PubMed: 11591669. PubMed DOI PMC

Saito F, Hori K, Kanda M, Kurotsu T, Saito Y (1994) Entire nucleotide-sequence for bacillus brevis nagano GRS2 gene encoding gramicidin-S synthetase-2 - a multifunctional peptide synthetase. J Biochem 116: 357-367. PubMed: 7822255. PubMed

Mootz HD, Marahiel MA (1997) The tyrocidine biosynthesis operon of Bacillus brevis: Complete nucleotide sequence and biochemical characterization of functional internal adenylation domains. J Bacteriol 179: 6843-6850. PubMed: 9352938. PubMed PMC

Samel SA, Wagner B, Marahiel MA, Essen LO (2006) The thioesterase domain of the fengycin biosynthesis cluster: A structural base for the macrocyclization of a non-ribosomal lipopeptide. J Mol Biol 359: 876-889. doi:10.1016/j.jmb.2006.03.062. PubMed: 16697411. PubMed DOI

Tsuge K, Ano T, Hirai M, Nakamura Y, Shoda M (1999) The genes degQ, pps, and lpa-8 (sfp) are responsible for conversion of Bacillus subtilis 168 to plipastatin production. Antimicrob Agents Chemother 43: 2183-2192. PubMed: 10471562. PubMed PMC

Kawasaki T, Sakurai F, Nagatsuka SY, Hayakawa Y (2009) Prodigiosin biosynthesis gene cluster in the roseophilin producer Streptomyces griseoviridis . J Antibiot (Tokyo) 62: 271-276. doi:10.1038/ja.2009.27. PubMed: 19329986. PubMed DOI

Kim D, Park YK, Lee JS, Kim JF, Jeong H et al. (2006) Analysis of a prodigiosin biosynthetic gene cluster from the marine bacterium Hahella chejuensis KCTC 2396. J Microbiol Biotechnol 16: 1912-1918.

Meiser P, Weissman KJ, Bode HB, Krug D, Dickschat JS et al. (2008) DKxanthene biosynthesis - Understanding the basis for diversity-oriented secondary metabolism. Chem Biol 15: 771-781. doi:10.1016/j.chembiol.2008.06.005. PubMed: 18721748. PubMed DOI

Zhang XJ, Parry RJ (2007) Cloning and characterization of the pyrrolomycin biosynthetic gene clusters from Actinosporangium vitaminophilum ATCC 31673 and Streptomyces sp strain UC 11065. Antimicrob Agents Chemother 51: 946-957. doi:10.1128/AAC.01214-06. PubMed: 17158935. PubMed DOI PMC

Maharjan S, Aryal N, Bhattarai S, Koju D, Lamichhane J et al. (2012) Biosynthesis of the nargenicin A(1) pyrrole moiety from Nocardia sp CS682. Appl Microbiol Biotechnol 93: 687-696. doi:10.1007/s00253-011-3567-x. PubMed: 21927992. PubMed DOI

Li CX, Roege KE, Kelly WL (2009) Analysis of the Indanomycin Biosynthetic Gene Cluster from Streptomyces antibioticus NRRL 8167. Chembiochem 10: 1064-1072. doi:10.1002/cbic.200800822. PubMed: 19301315. PubMed DOI

Wu QL, Liang JD, Lin SJ, Zhou XF, Bai LQ et al. (2011) Characterization of the biosynthesis gene cluster for the pyrrole polyether antibiotic calcimycin (A23187) in Streptomyces chartreusis NRRL 3882. Antimicrob Agents Chemother 55: 974-982. doi:10.1128/AAC.01130-10. PubMed: 21173184. PubMed DOI PMC

Méjean A, Mann S, Maldiney T, Vassiliadis G, Lequin O et al. (2009) Evidence that biosynthesis of the neurotoxic alkaloids anatoxin-a and homoanatoxin-a in the cyanobacterium Oscillatoria PCC 6506 occurs on a modular polyketide synthase initiated by L-proline. J Am Chem Soc 131: 7512-7513. doi:10.1021/ja9024353. PubMed: 19489636. PubMed DOI

Rantala-Ylinen A, Känä S, Wang H, Rouhiainen L, Wahlsten M et al. (2011) Anatoxin-a synthetase gene cluster of the cyanobacterium Anabaena sp strain 37 and molecular methods to detect potential producers. Appl Environ Microbiol 77: 7271-7278. doi:10.1128/AEM.06022-11. PubMed: 21873484. PubMed DOI PMC

Becker JE, Moore RE, Moore BS (2004) Cloning, sequencing, and biochemical characterization of the nostocyclopeptide biosynthetic gene cluster: molecular basis for imine macrocyclization. Gene 325: 35-42. doi:10.1016/j.gene.2003.09.034. PubMed: 14697508. PubMed DOI

Conti E, Stachelhaus T, Marahiel MA, Brick P (1997) Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S. EMBO J 16: 4174-4183. doi:10.1093/emboj/16.14.4174. PubMed: 9250661. PubMed DOI PMC

Stachelhaus T, Mootz HD, Marahiel MA (1999) The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases. Chem Biol 6: 493-505. doi:10.1016/S1074-5521(99)80082-9. PubMed: 10421756. PubMed DOI

Challis GL, Ravel J, Townsend CA (2000) Predictive, structure-based model of amino acid recognition by nonribosomal peptide synthetase adenylation domains. Chem Biol 7: 211-224. doi:10.1016/S1074-5521(00)00091-0. PubMed: 10712928. PubMed DOI

Kurmayer R, Christiansen G, Gumpenberger M, Fastner J (2005) Genetic identification of microcystin ecotypes in toxic cyanobacteria of the genus Planktothrix . Microbiology 151: 1525-1533. doi:10.1099/mic.0.27779-0. PubMed: 15870462. PubMed DOI

Najmanová L, Kutejová E, Kadlec J, Polan M, Olšovská J et al. (2013) Characterization of N-demethyl lincosamide methyltransferases LmbJ and CcbJ ChemBioChem In press. doi:10.1002/cbic.201300389. PubMed DOI

Magerlein BJ (1971) Modification of lincomycin. In: Perlman D. Structure-activity relationships among the semisynthetic antibiotics. New York: Academic Press; pp. 600-651.

Ulanova D, Novotná J, Smutná Y, Kameník Z, Gažák R et al. (2010) Mutasynthesis of lincomycin derivatives with activity against drug-resistant staphylococci. Antimicrob Agents Chemother 54: 927-930. doi:10.1128/AAC.00918-09. PubMed: 19917754. PubMed DOI PMC

Shaw-Reid CA, Kelleher NL, Losey HC, Gehring AM, Berg C et al. (1999) Assembly line enzymology by multimodular nonribosomal peptide synthetases: the thioesterase domain of E. coli EntF catalyzes both elongation and cyclolactonization. Chem Biol 6: 385-400. doi:10.1016/S1074-5521(99)80050-7. PubMed: 10375542. PubMed DOI

Lautru S, Challis GL (2004) Substrate recognition by nonribosomal peptide synthetase multi-enzymes. Microbiology 150: 1629-1636. doi:10.1099/mic.0.26837-0. PubMed: 15184549. PubMed DOI

Bushley KE, Ripoll DR, Turgeon BG (2008) Module evolution and substrate specificity of fungal nonribosomal peptide synthetases involved in siderophore biosynthesis. BMC Evol Biol 8: 328-. PubMed: 19055762. PubMed PMC

Yonus H, Neumann P, Zimmermann S, May JJ, Marahiel MA et al. (2008) Crystal structure of DltA. Implications for the reaction mechanism of non-ribosomal peptide synthetase adenylation domains. J Biol Chem 283: 32484-32491. doi:10.1074/jbc.M800557200. PubMed: 18784082. PubMed DOI

Crüsemann M, Kohlhaas C, Piel J (2013) Evolution-guided engineering of nonribosomal peptide synthetase adenylation domains. Chem Sci 4: 1041-1045. doi:10.1039/c2sc21722h. DOI

Milo R, Last RL (2012) Achieving diversity in the face of constraints: Lessons from metabolism. Science 336: 1663-1667. doi:10.1126/science.1217665. PubMed: 22745419. PubMed DOI

Liscombe DK, Louie GV, Noel JP (2012) Architectures, mechanisms and molecular evolution of natural product methyltransferases. Nat Prod Rep 29: 1238-1250. doi:10.1039/c2np20029e. PubMed: 22850796. PubMed DOI

Fischbach MA, Walsh CT, Clardy J (2008) The evolution of gene collectives: How natural selection drives chemical innovation. Proc Natl Acad Sci U S A 105: 4601-4608. doi:10.1073/pnas.0709132105. PubMed: 18216259. PubMed DOI PMC

O'Brien PJ, Herschlag D (1999) Catalytic promiscuity and the evolution of new enzymatic activities. Chem Biol 6: R91-R105. doi:10.1016/S1074-5521(99)80021-0. PubMed: 10099128. PubMed DOI

Gerratana B (2012) Biosynthesis, synthesis, and biological activities of pyrrolobenzodiazepines. Med Res Rev 32: 254-293. doi:10.1002/med.20212. PubMed: 20544978. PubMed DOI PMC

Kyselková M, Janata J, Ságová-Marečková M, Kopecký J (2010) Subunit-subunit interactions are weakened in mutant forms of acetohydroxy acid synthase insensitive to valine inhibition. Arch Microbiol 192: 195-200. doi:10.1007/s00203-010-0545-0. PubMed: 20107768. PubMed DOI

Geneious version 5.5.6 created by Biomatters. Available from ​ http://www.geneious.com/.

Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol Biol Evol 30: 772-780. doi:10.1093/molbev/mst010. PubMed: 23329690. PubMed DOI PMC

Tamura K, Peterson D, Peterson N, Stecher G, Nei M et al. (2011) MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 2731-2739. doi:10.1093/molbev/msr121. PubMed: 21546353. PubMed DOI PMC

Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA et al. (2007) Clustal W and clustal X version 2.0. Bioinformatics 23: 2947-2948. doi:10.1093/bioinformatics/btm404. PubMed: 17846036. PubMed DOI

Schwede T, Kopp J, Guex N, Peitsch MC (2003) SWISS-MODEL: An automated protein homology-modeling server. Nucleic Acids Res 31: 3381-3385. doi:10.1093/nar/gkg520. PubMed: 12824332. PubMed DOI PMC

Schrödinger LLC (2010) The PyMOL molecular graphics system, version 1.3.

Case DA, Cheatham TE, Darden T, Gohlke H, Luo R et al. (2005) The Amber biomolecular simulation programs. J Comput Chem 26: 1668-1688. doi:10.1002/jcc.20290. PubMed: 16200636. PubMed DOI PMC

Hornak V, Abel R, Okur A, Strockbine B, Roitberg A et al. (2006) Comparison of multiple amber force fields and development of improved protein backbone parameters. Proteins 65: 712-725. doi:10.1002/prot.21123. PubMed: 16981200. PubMed DOI PMC

Popov AV, Vorobjev YN, Zharkov DO (2013) MDTRA: A molecular dynamics trajectory analyzer with a graphical user interface. J Comput Chem 34: 319-325. doi:10.1002/jcc.23135. PubMed: 23047307. PubMed DOI

Evolution-guided adaptation of an adenylation domain substrate specificity to an unusual amino acid

Elucidation of salicylate attachment in celesticetin biosynthesis opens the door to create a library of more efficient hybrid lincosamide antibiotics

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?

Deacetylation of mycothiol-derived 'waste product' triggers the last biosynthetic steps of lincosamide antibiotics

Lincosamide synthetase--a unique condensation system combining elements of nonribosomal peptide synthetase and mycothiol metabolism

Sequence analysis of porothramycin biosynthetic gene cluster