Structurally different and functionally diverse natural compounds - antitumour agents pyrrolo[1,4]benzodiazepines, bacterial hormone hormaomycin, and lincosamide antibiotic lincomycin - share a common building unit, 4-alkyl-L-proline derivative (APD). APDs arise from L-tyrosine through a special biosynthetic pathway. Its generally accepted scheme, however, did not comply with current state of knowledge. Based on gene inactivation experiments and in vitro functional tests with recombinant enzymes, we designed a new APD biosynthetic scheme for the model of lincomycin biosynthesis. In the new scheme at least one characteristic in each of five final biosynthetic steps has been changed: the order of reactions, assignment of enzymes and/or reaction mechanisms. First, we demonstrate that LmbW methylates a different substrate than previously assumed. Second, we propose a unique reaction mechanism for the next step, in which a putative γ-glutamyltransferase LmbA indirectly cleaves off the oxalyl residue by transient attachment of glutamate to LmbW product. This unprecedented mechanism would represent the first example of the C-C bond cleavage catalyzed by a γ-glutamyltransferase, i.e., an enzyme that appears unsuitable for such activity. Finally, the inactivation experiments show that LmbX is an isomerase indicating that it transforms its substrate into a compound suitable for reduction by LmbY, thereby facilitating its subsequent complete conversion to APD 4-propyl-L-proline. Elucidation of the APD biosynthesis has long time resisted mainly due to the apparent absence of relevant C-C bond cleaving enzymatic activity. Our proposal aims to unblock this situation not only for lincomycin biosynthesis, but generally for all above mentioned groups of bioactive natural products with biotechnological potential.

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

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Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., et al. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. PubMed PMC

Blankenfeldt W., Kuzin A. P., Skarina T., Korniyenko Y., Tong L., Bayer P., et al. (2004). Structure and function of the phenazine biosynthetic protein PhzF from PubMed DOI PMC

Brahme N. M., Gonzalez J. E., Rolls J. P., Hessler E. J., Mizsak S., Hurley L. H. (1984). Biosynthesis of the lincomycins. 1. Studies using stable isotopes on the biosynthesis of the propyl- and ethyl-L-hygric acid moieties of lincomycins A and B. DOI

Braun S. D., Hofmann J., Wensing A., Ullrich M., Weingart H., Völksch B., et al. (2010). Identification of the biosynthetic gene cluster for 3-methylarginine, a toxin produced by PubMed DOI PMC

Castellano I., Merlino A. (2012). γ-Glutamyltranspeptidases: sequence, structure, biochemical properties, and biotechnological applications. PubMed DOI PMC

Colabroy K. L., Hackett W. T., Markham A. J., Rosenberg J., Cohen D. E., Jacobson A. (2008). Biochemical characterization of L-DOPA 2,3-dioxygenase, a single-domain type I extradiol dioxygenase from lincomycin biosynthesis. PubMed DOI

Colabroy K. L., Smith I. R., Vlahos A. H., Markham A. J., Jakubik M. E. (2014). Defining a kinetic mechanism for L-DOPA 2,3 dioxygenase, a single-domain type I extradiol dioxygenase from PubMed DOI

Connor K. L., Colabroy K. L., Gerratana B. (2011). A heme peroxidase with a functional role as an L-tyrosine hydroxylase in the biosynthesis of anthramycin. PubMed DOI PMC

Gauze G. F., Preobrazhenskaia T. P., Ivanitskaia L. P., Sveshnikova M. A. (1969). Production of the antibiotic sibiromycin by the PubMed

Gerratana B. (2012). Biosynthesis, synthesis, and biological activities of pyrrolobenzodiazepines. PubMed DOI PMC

Gust B., Chandra G., Jakimowicz D., Yuqing T., Bruton C. J., Chater K. F. (2004). Lambda red-mediated genetic manipulation of antibiotic-producing PubMed DOI

Hoeksema H., Bannister B., Birkenmeyer R., Kagan F., Magerlein B. J., MacKellar F. A., et al. (1964). Chemical studies on lincomycin. I. The structure of lincomycin. DOI

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. PubMed DOI

Hong H.-J., Hutchings M. I., Hill L. M., Buttner M. J. (2005). The role of the novel Fem protein VanK in vancomycin resistance in PubMed DOI

Hu Y., Phelan V., Ntai I., Farnet C. M., Zazopoulos E., Bachmann B. O. (2007). Benzodiazepine biosynthesis in PubMed DOI

Huang Y. T., Lyu S. Y., Chuang P. H., Hsu N. S., Li Y. S., Chan H. C., et al. (2009). In vitro characterization of enzymes involved in the synthesis of nonproteinogenic residue (2S,3S)-β-methylphenylalanine in glycopeptide antibiotic mannopeptimycin. PubMed DOI

Hurley L. H., Lasswell W. L., Ostrander J. M., Parry R. (1979). Pyrrolo[1,4]benzodiazepine antibiotics. Biosynthetic conversion of tyrosine to the C2-and C3-proline moieties of anthramycin, tomaymycin, and sibiromycin. PubMed DOI

Janata J., Kadlcik S., Koberska M., Ulanova D., Kamenik Z., Novak P., et al. (2015). Lincosamide synthetase-a unique condensation system combining elements of nonribosomal peptide synthetase and mycothiol metabolism. PubMed DOI PMC

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. PubMed DOI PMC

Kamenik Z., Kadlcik S., Radojevic B., Jiraskova P., Kuzma M., Gazak R., et al. (2016). Deacetylation of mycothiol-derived ‘waste product’triggers the last biosynthetic steps of lincosamide antibiotics. 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. PubMed DOI

Kariyone K., Yazawa H., Kohsaka M. (1971). The structures of tomaymycin and oxotomaymycin. DOI

Kieser T., Bibb M., Buttner M., Chater K., Hopwood D. (2000).

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 PubMed DOI

Kuo M., Yurek D., Coats J., Chung S., Li G. (1992). Isolation and identification of 3-propylidene-Δ1-pyrroline-5-carboxylic acid, a biosynthetic precursor of lincomycin. PubMed DOI

Leimgruber W., Batcho A., Schenker F. (1965). The structure of anthramycin. PubMed DOI

Li H., Graupner M., Xu H., White R. H. (2003). CofE catalyzes the addition of two glutamates to F420-0 in F420 coenzyme biosynthesis in PubMed DOI

Li W., Chou S. C., Khullar A., Gerratana B. (2009a). Cloning and characterization of the biosynthetic gene cluster for tomaymycin, an SJG-136 monomeric analog. PubMed DOI PMC

Li W., Khullar A., Chou S., Sacramo A., Gerratana B. (2009b). Biosynthesis of sibiromycin, a potent antitumor antibiotic. PubMed DOI PMC

Lin C. I., Sasaki E., Zhong A. S., Liu H. W. (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. PubMed DOI PMC

Magerlein B. J. (1977). “Modification of lincomycin,” in

Najmanova L., Ulanova D., Jelinkova M., Kamenik Z., Kettnerova E., Koberska M., et al. (2014). Sequence analysis of porothramycin biosynthetic gene cluster. PubMed DOI PMC

Neusser D., Schmidt H., Spizek J., Novotna J., Peschke U., Kaschabeck S., et al. (1998). The genes lmbB1 and lmbB2 of PubMed DOI

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. PubMed DOI

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. PubMed DOI PMC

Olsovska J., Jelinkova M., Man P., Koberska M., Janata J., Flieger M. (2007). High-throughput quantification of lincomycin traces in fermentation broth of genetically modified PubMed DOI

Pang A.-P., Du L., Lin C.-Y., Qiao J., Zhao G.-R. (2015). Co-overexpression of lmbW and metK led to increased lincomycin A production and decreased byproduct lincomycin B content in an industrial strain of PubMed DOI

Parsons J. F., Song F., Parsons L., Calabrese K., Eisenstein E., Ladner J. E. (2004). Structure and function of the phenazine biosynthesis protein PhzF from PubMed DOI

Peschke U., Schmidt H., Zhang H. Z., Piepersberg W. (1995). Molecular characterization of the lincomycin-production gene cluster of PubMed DOI

Rössner E., Zeeck A., König W. A. (1990). Aufklärung der struktur von hormaomycin. DOI

Saha S., Li W., Gerratana B., Rokita S. E. (2015). Identification of the dioxygenase-generated intermediate formed during biosynthesis of the dihydropyrrole moiety common to anthramycin and sibiromycin. PubMed DOI PMC

Sasaki E., Lin C. I., Lin K. Y., Liu H. W. (2012). Construction of the octose 8-phosphate intermediate in lincomycin A biosynthesis: characterization of the reactions catalyzed by LmbR and LmbN. PubMed DOI PMC

Schauer N. L., Ferry J. G., Honek J. F., Orme-Johnson W. H., Walsh C. (1986). Mechanistic studies of the coenzyme F420-reducing formate dehydrogenase from PubMed DOI

Schneditz G., Rentner J., Roier S., Pletz J., Herzog K. A., Bücker R., et al. (2014). Enterotoxicity of a nonribosomal peptide causes antibiotic-associated colitis. PubMed DOI PMC

Tsunakawa M., Kamei H., Konishi M., Miyaki T., Oki T., Kawaguchi H. (1988). Porothramycin, a new antibiotic of the anthramycin group: production, isolation, structure and biological activity. 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. PubMed DOI PMC

Zhao Q., Wang M., Xu D., Zhang Q., Liu W. (2015). Metabolic coupling of two small-molecule thiols programs the biosynthesis of lincomycin A. PubMed DOI

Discovery of nostatin A, an azole-containing proteusin with prominent cytostatic and pro-apoptotic activity

C-C bond cleavage in biosynthesis of 4-alkyl-L-proline precursors of lincomycin and anthramycin cannot precede C-methylation

Novel pathway of 3-hydroxyanthranilic acid formation in limazepine biosynthesis reveals evolutionary relation between phenazines and pyrrolobenzodiazepines

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