In the biosynthesis of lincosamide antibiotics lincomycin and celesticetin, the amino acid and amino sugar units are linked by an amide bond. The respective condensing enzyme lincosamide synthetase (LS) is expected to be an unusual system combining nonribosomal peptide synthetase (NRPS) components with so far unknown amino sugar related activities. The biosynthetic gene cluster of celesticetin was sequenced and compared to the lincomycin one revealing putative LS coding ORFs shared in both clusters. Based on a bioassay and production profiles of S. lincolnensis strains with individually deleted putative LS coding genes, the proteins LmbC, D, E, F and V were assigned to LS function. Moreover, the newly recognized N-terminal domain of LmbN (LmbN-CP) was also assigned to LS as a NRPS carrier protein (CP). Surprisingly, the homologous CP coding sequence in celesticetin cluster is part of ccbZ gene adjacent to ccbN, the counterpart of lmbN, suggesting the gene rearrangement, evident also from still active internal translation start in lmbN, and indicating the direction of lincosamide biosynthesis evolution. The in vitro test with LmbN-CP, LmbC and the newly identified S. lincolnensis phosphopantetheinyl transferase Slp, confirmed the cooperation of the previously characterized NRPS A-domain LmbC with a holo-LmbN-CP in activation of a 4-propyl-L-proline precursor of lincomycin. This result completed the functional characterization of LS subunits resembling NRPS initiation module. Two of the four remaining putative LS subunits, LmbE/CcbE and LmbV/CcbV, exhibit low but significant homology to enzymes from the metabolism of mycothiol, the NRPS-independent system processing the amino sugar and amino acid units. The functions of particular LS subunits as well as cooperation of both NRPS-based and NRPS-independent LS blocks are discussed. The described condensing enzyme represents a unique hybrid system with overall composition quite dissimilar to any other known enzyme system.

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

Institute of Microbiology Academy of Sciences of the Czech Republic v v i Prague Czech Republic; Oceanography Section Science Research Center Kochi University IMT MEXT Kohasu Oko cho Nankoku Kochi 783 8505 Japan

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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. PubMed

Piepersberg W (1995) Streptomycin and related aminoglycosides. Biotechnology (Reading, Mass) 28: 531–570. PubMed

Chung S- T, Manis JJ, McWethy SJ, Patt TE, Witz DF, Wolf HJ, et al. (1997) Fermentation, biosynthesis and molecular genetics of lincomycin In: Strohl WR, editor. Biotechnology of Antibiotics. 2 ed New York: Dekker; pp. 165–186.

Kadlcik S, Kucera T, Chalupska D, Gazak R, Koberska M, Ulanova D, et al. (2013) Adaptation of an L-Proline Adenylation Domain to Use 4-Propyl-L-Proline in the Evolution of Lincosamide Biosynthesis. PLoS One 8(12) 10.1371/journal.pone.0084902 PubMed DOI PMC

Marahiel M, Essen LO (2009) Nonribosomal peptide synthetases: mechanistic and structural aspects of essential domains. Methods Enzymol 458: 337–351. 10.1016/S0076-6879(09)04813-7 PubMed DOI

Koberska M, Kopecky J, Olsovska J, Jelinkova M, Ulanova D, Man P, 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. 10.1007/s12223-008-0060-8 PubMed DOI

Wilkinson B, Micklefield J (2009) Biosynthesis of nonribosomal peptide precursors. Methods Enzymol 458: 353–378. 10.1016/S0076-6879(09)04814-9 PubMed DOI

Maruyama C, Toyoda J, Kato Y, Izumikawa M, Takagi M, Shin-ya K, et al. (2012) A stand-alone adenylation domain forms amide bonds in streptothricin biosynthesis. Nat Chem Biol 8: 791–797. 10.1038/nchembio.1040 PubMed DOI

Newton GL, Buchmeier N, Fahey RC (2008) Biosynthesis and functions of mycothiol, the unique protective thiol of Actinobacteria. Microbiol Mol Biol Rev 72: 471–494. 10.1128/MMBR.00008-08 PubMed DOI PMC

Brahme NM, Gonzalez J, Mizsak S, Rolls J, Hessler E, Hurley L (1984) Biosynthesis of the lincomycins. 2. Studies using stable isotopes on the biosynthesis of methylthiolincosaminide moiety of lincomycin A. J Am Chem Soc 106: 7878–7883.

Najmanova L, Kutejova E, Kadlec J, Polan M, Olsovska J, Benada O, et al. (2013) Characterization of N-Demethyllincosamide Methyltransferases LmbJ and CcbJ. Chembiochem 14: 2259–2262. 10.1002/cbic.201300389 PubMed DOI

Bauer J, Ondrovicova G, Najmanova L, Pevala V, Kamenik Z, Kostan J, et al. (2014) Structure and possible mechanism of the CcbJ methyltransferase from Streptomyces caelestis . Acta Crystallogr D Biol Crystallogr 70: 943–957. 10.1107/S139900471303397X PubMed DOI

Lin CI, Sasaki E, Zhong AS, Liu HW (2014) In Vitro Characterization of LmbK and LmbO: Identification of GDP-D-erythro-alpha-D-gluco-octose as a Key Intermediate in Lincomycin A Biosynthesis. J Am Chem Soc 136: 906–909. 10.1021/ja412194w PubMed DOI PMC

Sasaki E, Lin CI, Lin KY, Liu HW (2012) Construction of the Octose 8-Phosphate Intermediate in Lincomycin A Biosynthesis: Characterization of the Reactions Catalyzed by LmbR and LmbN. J Am Chem Soc 134: 17432–17435. 10.1021/ja308221z PubMed DOI PMC

Gust B, Challis GL, Fowler K, Kieser T, Chater KF (2003) PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc Natl Acad Sci USA 100: 1541–1546. PubMed PMC

Atlas RM (2004) Handbook of microbiological media; Atlas RM, editor: CRC press.

Kieser T, Bibb M, Buttner M, Chater K, Hopwood D (2000) Practical Streptomyces Genetics. Norwich, UK: The John Innes Foundation; 613 p.

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

Kamenik Z, Kopecky J, Mareckova M, Ulanova D, Novotna J, Pospisil S, et al. (2009) HPLC-fluorescence detection method for determination of key intermediates of the lincomycin biosynthesis in fermentation broth. Anal Bioanal Chem 393: 1779–1787. 10.1007/s00216-009-2605-3 PubMed DOI

Ulanova D, Novotna J, Smutna Y, Kamenik Z, Gazak R, Sulc M, et al. (2010) Mutasynthesis of Lincomycin Derivatives with Activity against Drug-Resistant Staphylococci. Antimicrob Agents Chemother 54: 927–930. 10.1128/AAC.00918-09 PubMed DOI PMC

Hong H- J, Hutchings MI, Hill LM, Buttner MJ (2005) The role of the novel Fem protein VanK in vancomycin resistance in Streptomyces coelicolor . J Biol Chem 280: 13055–13061. PubMed

Marchler-Bauer A, Lu S, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese-Scott C, et al. (2011) CDD: a Conserved Domain Database for the functional annotation of proteins. Nucleic Acids Res 39: D225–D229. 10.1093/nar/gkq1189 PubMed DOI PMC

Hu Y, Phelan V, Ntai I, Farnet CM, Zazopoulos E, Bachmann BO (2007) Benzodiazepine biosynthesis in Streptomyces refuineus . Chem Biol 14: 691–701. PubMed

Li W, Khullar A, Chou S, Sacramo A, Gerratana B (2009) Biosynthesis of Sibiromycin, a Potent Antitumor Antibiotic. Appl Environ Microbiol 75: 2869–2878. 10.1128/AEM.02326-08 PubMed DOI PMC

Novotna J, Honzatko A, Bednar P, Kopecky J, Janata J, Spizek J (2004) L-3,4-Dihydroxyphenyl alanine-extradiol cleavage is followed by intramolecular cyclization in lincomycin biosynthesis. Eur J Biochem 271: 3678–3683. PubMed

Novotna J, Olsovska J, Novak P, Mojzes P, Chaloupkova R, Kamenik Z, et al. (2013) Lincomycin Biosynthesis Involves a Tyrosine Hydroxylating Heme Protein of an Unusual Enzyme Family. PLoS One 8(12) 10.1371/journal.pone.0079974 PubMed DOI PMC

Dangel V, Eustaquio AS, Gust B, Heide L (2008) novE and novG act as positive regulators of novobiocin biosynthesis. Arch Microbiol 190: 509–519. 10.1007/s00203-008-0396-0 PubMed DOI

Chen L, Wang ZL, Zhao QF, Gao SH, Ye RF (2011) Functional determination of the regulatory gene lmbU in lincomycin biosynthesis gene cluster. Chemistry & Bioengineering 28: 37–41.

Ferrer S, Martí S, Moliner V, Tuñón I, Bertrán J (2012) Understanding the different activities of highly promiscuous MbtI by computational methods. Phys Chem Chem Phys 14: 3482–3489. 10.1039/c2cp23149b PubMed DOI

Kerbarh O, Ciulli A, Howard NI, Abell C (2005) Salicylate biosynthesis: overexpression, purification, and characterization of Irp9, a bifunctional salicylate synthase from Yersinia enterocolitica . J Bacteriol 187: 5061–5066. PubMed PMC

Ishiyama D, Vujaklija D, Davies J (2004) Novel pathway of salicylate degradation by Streptomyces sp. strain WA46. Appl Env Microbiol 70: 1297–1306. PubMed PMC

Stachelhaus T, Mootz HD, Bergendahl V, Marahiel MA (1998) Peptide bond formation in nonribosomal peptide biosynthesis catalytic role of the condensation domain. J Biol Chem 273: 22773–22781. PubMed

Lambalot RH, Gehring AM, Flugel RS, Zuber P, LaCelle M, Marahiel MA, et al. (1996) A new enzyme superfamily—the phosphopantetheinyl transferases. Chem Biol 3: 923–936. PubMed

Reuter K, Mofid MR, Marahiel MA, Ficner R (1999) Crystal structure of the surfactin synthetase‐activating enzyme Sfp: a prototype of the 4′‐phosphopantetheinyl transferase superfamily. EMBO J 18: 6823–6831. PubMed PMC

Sánchez C, Du L, Edwards DJ, Toney MD, Shen B (2001) Cloning and characterization of a phosphopantetheinyl transferase from Streptomyces verticillus ATCC15003, the producer of the hybrid peptide—polyketide antitumor drug bleomycin. Chem Biol 8: 725–738. PubMed

Parris KD, Lin L, Tam A, Mathew R, Hixon J, Stahl M, et al. (2000) Crystal structures of substrate binding to Bacillus subtilis holo-(acyl carrier protein) synthase reveal a novel trimeric arrangement of molecules resulting in three active sites. Structure 8: 883–895. PubMed

Ku J, Mirmira RG, Liu L, Santi DV (1997) Expression of a functional non-ribosomal peptide synthetase module in Escherichia coli by coexpression with a phosphopantetheinyl transferase. Chem Biol 4: 203–207. PubMed

Quadri LE, Weinreb PH, Lei M, Nakano MM, Zuber P, Walsh CT (1998) Characterization of Sfp, a Bacillus subtilis phosphopantetheinyl transferase for peptidyl carrier protein domains in peptide synthetases. Biochemistry 37: 1585–1595. 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. PubMed

Méjean A, Mann S, Vassiliadis G, Lombard Brr, Loew D, Ploux O (2009) 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. PubMed

Gehring AM, Bradley KA, Walsh CT (1997) Enterobactin biosynthesis in Escherichia coli: isochorismate lyase (EntB) is a bifunctional enzyme that is phosphopantetheinylated by EntD and then acylated by EntE using ATP and 2, 3-dihydroxybenzoate. Biochemistry 36: 8495–8503. PubMed

Grammel N, Pankevych K, Demydchuk J, Lambrecht K, Saluz HP, Krügel H (2002) A β‐lysine adenylating enzyme and a β‐lysine binding protein involved in poly β‐lysine chain assembly in nourseothricin synthesis in Streptomyces noursei . Eur J Biochem 269: 347–357. PubMed

Harris AK, Williamson NR, Slater H, Cox A, Abbasi S, Foulds I, et al. (2004) The Serratia gene cluster encoding biosynthesis of the red antibiotic, prodigiosin, shows species-and strain-dependent genome context variation. Microbiol 150: 3547–3560. PubMed

Stanley AE, Walton LJ, Zerikly MK, Corre C, Challis GL (2006) Elucidation of the Streptomyces coelicolor pathway to 4-methoxy-2, 2′-bipyrrole-5-carboxaldehyde, an intermediate in prodiginine biosynthesis. Chem Commun: 3981–3983. PubMed

Finking R, Solsbacher J, Konz D, Schobert M, Schäfer A, Jahn D, et al. (2002) Characterization of a new type of phosphopantetheinyl transferase for fatty acid and siderophore synthesis in Pseudomonas aeruginosa . J Biol Chem 277: 50293–50302. PubMed

Bunet R, Riclea R, Laureti L, Hôtel L, Paris C, Girardet JM, et al. (2014) A Single Sfp-Type Phosphopantetheinyl Transferase Plays a Major Role in the Biosynthesis of PKS and NRPS Derived Metabolites in Streptomyces ambofaciens ATCC23877. PLoS One 9: e87607 10.1371/journal.pone.0087607 PubMed DOI PMC

Copp J, Neilan B (2006) The phosphopantetheinyl transferase superfamily: phylogenetic analysis and functional implications in cyanobacteria. Appl Environ Microbiol 72: 2298–2305. PubMed PMC

Newton GL, Buchmeier N, Fahey RC (2008) Biosynthesis and functions of mycothiol, the unique protective thiol of Actinobacteria. Microbiol Mol Biol Rev 72: 471–494. 10.1128/MMBR.00008-08 PubMed DOI PMC

Jothivasan VK, Hamilton CJ (2008) Mycothiol: synthesis, biosynthesis and biological functions of the major low molecular weight thiol in actinomycetes. Nat Prod Rep 25: 1091–1117. 10.1039/b616489g PubMed DOI

Gaballa A, Newton GL, Antelmann H, Parsonage D, Upton H, Rawat M, et al. (2010) Biosynthesis and functions of bacillithiol, a major low-molecular-weight thiol in Bacilli. Proc Natl Acad Sci 107: 6482–6486. 10.1073/pnas.1000928107 PubMed DOI PMC

Sareen D, Steffek M, Newton GL, Fahey RC (2002) ATP-dependent L-cysteine: 1D-myo-inosityl 2-amino-2-deoxy-α-D-glucopyranoside ligase, mycothiol biosynthesis enzyme MshC, is related to class I cysteinyl-tRNA synthetases. Biochemistry 41: 6885–6890. PubMed

Ángel Rubio M, Barrado P, Carlos Espinosa J, Jiménez A, Lobato MaF (2004) The pur6 gene of the puromycin biosynthetic gene cluster from Streptomyces alboniger encodes a tyrosinyl-aminonucleoside synthetase. FEBS Lett 577: 371–375. PubMed

Newton GL, Arnold K, Price MS, Sherrill C, Delcardayre SB, Aharonowitz Y, et al. (1996) Distribution of thiols in microorganisms: mycothiol is a major thiol in most actinomycetes. J Bacteriol 178: 1990–1995. PubMed PMC

Nakai T, Okada K, Akutsu S, Miyahara I, Kawaguchi S- I, Kato R, et al. (1999) Structure of Thermus thermophilus HB8 aspartate aminotransferase and its complex with maleate. Biochemistry 38: 2413–2424. PubMed

Newton GL, Leung SS, Wakabayashi JI, Rawat M, Fahey RC (2011) The DinB superfamily includes novel mycothiol, bacillithiol, and glutathione S-transferases. Biochemistry 50: 10751–10760. 10.1021/bi201460j PubMed DOI PMC

Wang R, Yin Y- J, Wang F, Li M, Feng J, Zhang HM, et al. (2007) Crystal structures and site-directed mutagenesis of a mycothiol-dependent enzyme reveal a novel folding and molecular basis for mycothiol-mediated maleylpyruvate isomerization. J Biol Chem 282: 16288–15294. PubMed

Zengliang W, Qunfei Z, Shuhong G, Changhua C, Wen L (2010) Construction of the New Genetic Manipulation Method of Streptomyces lincolnensis and lmbQ Gene Function Verification. Biotechnology Bulletin 11: 038.

Janata J, Najmanova L, Novotna J, Hola K, Felsberg J, Spizek J (2001) Putative lmbI and lmbH genes form a single lmbIH ORF in Streptomyces lincolnensis type strain ATCC 25466. Antonie Van Leeuwenhoek 79: 277–284. PubMed

Allali N, Afif H, Couturier M, Van Melderen L (2002) The highly conserved TldD and TldE proteins of Escherichia coli are involved in microcin B17 processing and in CcdA degradation. J Bacteriol 184: 3224–3231. PubMed PMC

Ahlert J, Shepard E, Lomovskaya N, Zazopoulos E, Staffa A, Bachmann BO, et al. (2002) The calicheamicin gene cluster and its iterative type I enediyne PKS. Science 297: 1173–1176. PubMed

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. 10.1128/AEM.02325-08 PubMed DOI PMC

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

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