Cysteamine Chemisorption at Mercury-Solution Interfaces in the Context of Redox and Microdissociation Equilibria.

The redox behavior and chemisorption of cysteamine (CA) at a charged mercury surface are described, with an emphasis on its acid-base properties supported by molecular dynamics and quantum mechanical calculations. It was found that CA forms chemisorbed layers on the surface of the mercury electrode. The formation of Hg-CA complexes is connected to mercury disproportionation, as reflected in peaks SII and SI at potentials higher than the electrode potential of zero charge (p.z.c.). Both the process of chemisorption of CA and its consequent redox transformation are proton-dependent. Also, depending on the protonation of CA, the formation of typical populations of chemisorbed conformers can be observed. In addition, cystamine (CA disulfide dimer) can be reduced on the mercury surface. Between the potentials of this reduction and peak SI, the p.z.c. of the electrode used can be found. Furthermore, CA can serve as an LMW catalyst for hydrogen evolution. The mechanistic insights presented here can be used for follow-up research on CA chemisorption and targeted modification of other metallic surfaces.

Structure-Function Analysis of the S-Glycosylation Reaction in the Biosynthesis of Lincosamide Antibiotics.

The S-glycosyltransferase LmbT, involved in the biosynthesis of lincomycin A, is the only known enzyme that catalyzes the enzymatic incorporation of rare amino acid L-ergothioneine (EGT) into secondary metabolites. Here, we show the structure and function analyses of LmbT. Our in vitro analysis of LmbT revealed that the enzyme shows promiscuous substrate specificity toward nitrogenous base moieties in the generation of unnatural nucleotide diphosphate (NDP)-D-α-D-lincosamides. Furthermore, the X-ray crystal structures of LmbT in its apo form and in complex with substrates indicated that the large conformational changes of the active site occur upon binding of the substrates, and that EGT is strictly recognized by salt-bridge and cation-π interactions with Arg260 and Trp101, respectively. The structure of LmbT in complex with its substrates, the docking model with the EGT-S-conjugated lincosamide, and the structure-based site-directed mutagenesis analysis revealed the structural details of the LmbT-catalyzed SN 2-like S-glycosylation reaction with EGT.

TldD/TldE peptidases and N-deacetylases: A structurally unique yet ubiquitous protein family in the microbial metabolism.

Here we provide a review on TldD/TldE family proteins, summarizing current knowledge and outlining further research perspectives. Despite being widely distributed in bacteria and archaea, TldD/TldE proteins have been escaping attention for a long time until several recent reports pointed to their unique features. Specifically, TldD/TldE generally act as peptidases, though some of them turned out to be N-deacetylases. Biological function of TldD/TldE has been extensively described in bacterial specialized metabolism, in which they participate in the biosynthesis of lincosamide antibiotics (as N-deacetylases), and in the biosynthesis of ribosomally synthesized and post-translationally modified bioactive peptides (as peptidases). These enzymes possess special position in the relevant biosynthesis since they convert non-bioactive intermediates into bioactive metabolites. Further, based on a recent study of Escherichia coli TldD/TldE, these heterodimeric metallopeptidases possess a new protein fold exhibiting several structural features with no precedent in the Protein Data Bank. The most interesting ones are structural elements forming metal-containing active site on the inner surface of the catalytically active subunit TldD, in which substrates bind through ß sheet interactions in the sequence-independent manner. It results in relaxed substrate specificity of TldD/TldE, which is counterbalanced by enclosing the active centre within the hollow core of the heterodimer and only appropriate substrates can entry through a narrow channel. Based on the published data, we hypothesize a yet unrecognized central metabolic function of TldD/TldE in the degradation of (partially) unfolded proteins, i.e., in protein quality control.

Beyond Self-Resistance: ABCF ATPase LmrC Is a Signal-Transducing Component of an Antibiotic-Driven Signaling Cascade Accelerating the Onset of Lincomycin Biosynthesis.

In natural environments, antibiotics are important means of interspecies competition. At subinhibitory concentrations, they act as cues or signals inducing antibiotic production; however, our knowledge of well-documented antibiotic-based sensing systems is limited. Here, for the soil actinobacterium Streptomyces lincolnensis, we describe a fundamentally new ribosome-mediated signaling cascade that accelerates the onset of lincomycin production in response to an external ribosome-targeting antibiotic to synchronize antibiotic production within the population. The entire cascade is encoded in the lincomycin biosynthetic gene cluster (BGC) and consists of three lincomycin resistance proteins in addition to the transcriptional regulator LmbU: a lincomycin transporter (LmrA), a 23S rRNA methyltransferase (LmrB), both of which confer high resistance, and an ATP-binding cassette family F (ABCF) ATPase, LmrC, which confers only moderate resistance but is essential for antibiotic-induced signal transduction. Specifically, antibiotic sensing occurs via ribosome-mediated attenuation, which activates LmrC production in response to lincosamide, streptogramin A, or pleuromutilin antibiotics. Then, ATPase activity of the ribosome-associated LmrC triggers the transcription of lmbU and consequently the expression of lincomycin BGC. Finally, the production of LmrC is downregulated by LmrA and LmrB, which reduces the amount of ribosome-bound antibiotic and thus fine-tunes the cascade. We propose that analogous ABCF-mediated signaling systems are relatively common because many ribosome-targeting antibiotic BGCs encode an ABCF protein accompanied by additional resistance protein(s) and transcriptional regulators. Moreover, we revealed that three of the eight coproduced ABCF proteins of S. lincolnensis are clindamycin responsive, suggesting that the ABCF-mediated antibiotic signaling may be a widely utilized tool for chemical communication. IMPORTANCE Resistance proteins are perceived as mechanisms protecting bacteria from the inhibitory effect of their produced antibiotics or antibiotics from competitors. Here, we report that antibiotic resistance proteins regulate lincomycin biosynthesis in response to subinhibitory concentrations of antibiotics. In particular, we show the dual character of the ABCF ATPase LmrC, which confers antibiotic resistance and simultaneously transduces a signal from ribosome-bound antibiotics to gene expression, where the 5' untranslated sequence upstream of its encoding gene functions as a primary antibiotic sensor. ABCF-mediated antibiotic signaling can in principle function not only in the induction of antibiotic biosynthesis but also in selective gene expression in response to any small molecules targeting the 50S ribosomal subunit, including clinically important antibiotics, to mediate intercellular antibiotic signaling and stress response induction. Moreover, the resistance-regulatory function of LmrC presented here for the first time unifies functionally inconsistent ABCF family members involving antibiotic resistance proteins and translational regulators.

The Oral Microbiome in Periodontal Health.

The estimation of oral microbiome (OM) taxonomic composition in periodontally healthy individuals can often be biased because the clinically periodontally healthy subjects for evaluation can already experience dysbiosis. Usually, they are included just based on the absence of clinical signs of periodontitis. Additionally, the age of subjects is used to be higher to correspond well with tested groups of patients with chronic periodontitis, a disorder typically associated with aging. However, the dysbiosis of the OM precedes the clinical signs of the disease by many months or even years. The absence of periodontal pockets thus does not necessarily mean also good periodontal health and the obtained image of "healthy OM" can be distorted.To overcome this bias, we taxonomically characterized the OM in almost a hundred young students of dentistry with precise oral hygiene and no signs of periodontal disease. We compared the results with the OM composition of older periodontally healthy individuals and also a group of patients with severe periodontitis (aggressive periodontitis according to former classification system). The clustering analysis revealed not only two compact clearly separated clusters corresponding to each state of health, but also a group of samples forming an overlap between both well-pronounced states. Additionally, in the cluster of periodontally healthy samples, few outliers with atypical OM and two major stomatotypes could be distinguished, differing in the prevalence and relative abundance of two main bacterial genera: Streptococcus and Veillonella. We hypothesize that the two stomatotypes could represent the microbial succession from periodontal health to starting dysbiosis. The old and young periodontally healthy subjects do not cluster separately but a trend of the OM in older subjects to periodontitis is visible. Several bacterial genera were identified to be typically more abundant in older periodontally healthy subjects.

R/G Value-A Numeric Index of Individual Periodontal Health and Oral Microbiome Dynamics.

The dysbiosis of oral microbiome (OM) precedes the clinical signs of periodontal disease. Its simple measure thus could indicate individuals at risk of periodontitis development; however, such a tool is still missing. Up to now, numerous microbial taxa were associated with periodontal health or periodontitis. The outputs of most studies could, nevertheless, be slightly biased from following two reasons: First, the healthy group is often characterized only by the absence of the disease, but the individuals could already suffer from dysbiosis without any visible signs. Second, the healthy/diseased OM characteristics are frequently determined based on average data obtained for whole groups of periodontally healthy persons versus patients. Especially in smaller sets of tested individuals the typical individual variability can thus complicate the unambiguous assignment of oral taxa to respective state of health. In this work the taxonomic composition of OM was evaluated for 20 periodontally healthy individuals and 15 patients with chronic periodontitis. The narrowed selection set of the most diseased patients (confirmed by clinical parameters) and the most distant group of healthy individuals with the lowest probability of dysbiosis was determined by clustering analysis and used for identification of marker taxa. Based on their representation in each individual oral cavity we proposed the numeric index of periodontal health called R/G value. Its diagnostic potential was further confirmed using independent set of 20 periodontally healthy individuals and 20 patients with periodontitis with 95 percent of samples assigned correctly. We also assessed the individual temporal OM dynamics in periodontal health and we compared it to periodontitis. We revealed that the taxonomic composition of the system changes dynamically but generally it ranges within values typical for periodontal health or transient state, but far from values typical for periodontitis. R/G value tool, formulated from individually evaluated data, allowed us to arrange individual OMs into a continuous series, instead of two distinct groups, thus mimicking the gradual transformation of a virtual person from periodontal health to disease. The application of R/G value index thus represents a very promising diagnostic tool for early prediction of persons at risk of developing periodontal disease.

N-Deacetylation in Lincosamide Biosynthesis Is Catalyzed by a TldD/PmbA Family Protein.

Lincosamides are clinically important antibiotics originally produced as microbial specialized metabolites. The complex biosynthesis of lincosamides is coupled to the metabolism of mycothiol as a sulfur donor. Here, we elucidated the N-deacetylation of the mycothiol-derived N-acetyl-l-cysteine residue of a lincosamide intermediate, which is comprised of an amino acid and an aminooctose connected via an amide bond. We purified this intermediate from the culture broth of a deletion mutant strain and tested it as a substrate of recombinant lincosamide biosynthetic proteins in the in vitro assays that were monitored via liquid chromatography-mass spectrometry. Our findings showed that the N-deacetylation reaction is catalyzed by CcbIH/CcbQ or LmbIH/LmbQ proteins in celesticetin and lincomycin biosynthesis, respectively. These are the first N-deacetylases from the TldD/PmbA protein family, from which otherwise only several proteases and peptidases were functionally characterized. Furthermore, we present a sequence similarity network of TldD/PmbA proteins, which suggests that the lincosamide N-deacetylases are unique among these widely distributed proteins.

Different Reaction Specificities of F420H2-Dependent Reductases Facilitate Pyrrolobenzodiazepines and Lincomycin To Fit Their Biological Targets.

Antitumor pyrrolobenzodiazepines (PBDs), lincosamide antibiotics, quorum-sensing molecule hormaomycin, and antimicrobial griselimycin are structurally and functionally diverse groups of actinobacterial metabolites. The common feature of these compounds is the incorporation of l-tyrosine- or l-leucine-derived 4-alkyl-l-proline derivatives (APDs) in their structures. Here, we report that the last reaction in the biosynthetic pathway of APDs, catalyzed by F420H2-dependent Apd6 reductases, contributes to the structural diversity of APD precursors. Specifically, the heterologous overproduction of six Apd6 enzymes demonstrated that Apd6 from the biosynthesis of PBDs and hormaomycin can reduce only an endocyclic imine double bond, whereas Apd6 LmbY and partially GriH from the biosyntheses of lincomycin and griselimycin, respectively, also reduce the more inert exocyclic double bond of the same 4-substituted Δ1-pyrroline-2-carboxylic acid substrate, making LmbY and GriH unusual, if not unique, among reductases. Furthermore, the differences in the reaction specificity of the Apd6 reductases determine the formation of the fully saturated APD moiety of lincomycin versus the unsaturated APD moiety of PBDs, providing molecules with optimal shapes to bind their distinct biological targets. Moreover, the Apd6 reductases establish the first F420H2-dependent enzymes from the luciferase-like hydride transferase protein superfamily in the biosynthesis of bioactive molecules. Finally, our bioinformatics analysis demonstrates that Apd6 and their homologues, widely distributed within several bacterial phyla, play a role in the formation of novel yet unknown natural products with incorporated l-proline-like precursors and likely in the microbial central metabolism.

New chalcone-sulfonamide hybrids exhibiting anticancer and antituberculosis activity.

New sulfonamides 5/6 derived from 4-methoxyacetophenone 1 were synthesized by N-sulfonation reaction of ammonia (3) and aminopyrimidinone (4) with its sulfonyl chloride derivative 2. Sulfonamides 5 and 6 were used as precursors of two new series of chalcones 8a-f and 9a-f, which were obtained through Claisen-Schmidt condensation with aromatic aldehydes 7a-f. Compounds 5/6, 8a-d, 8f, 9a-d, and 9f were screened by the US National Cancer Institute (NCI) at 10 µM against sixty different human cancer cell lines (one-dose trial). Chalcones 8b and 9b satisfied the pre-determined threshold inhibition criteria and were selected for screening at five different concentrations (100, 10, 1.0, 0.1, and 0.01 µM). Compound 8b exhibited remarkable GI50 values ranging from 0.57 to 12.4 µM, with cytotoxic effects being observed in almost all cases, especially against the cell lines K-562 of Leukemia and LOX IMVI of Melanoma with GI50 = 0.57 and 1.28 µM, respectively. Moreover, all compounds were screened against Mycobacterium tuberculosis H37Rv, chalcones 8a-c and 9a-c were the most active showing MIC values between 14 and 42 µM, and interestingly they were devoid of antibacterial activity against Mycobacterium smegmatis and Staphylococcus aureus. These antituberculosis hits showed however low selectivity, being equally inhibitory to M. tuberculosis and mammalian T3T cells. The chalcone-sulfonamide hybrids 8a-f and 9a-f resulted to be appealing cytotoxic agents with significant antituberculosis activity.

Suitability and setup of next-generation sequencing-based method for taxonomic characterization of aquatic microbial biofilm.

A robust and widely applicable method for sampling of aquatic microbial biofilm and further sample processing is presented. The method is based on next-generation sequencing of V4-V5 variable regions of 16S rRNA gene and further statistical analysis of sequencing data, which could be useful not only to investigate taxonomic composition of biofilm bacterial consortia but also to assess aquatic ecosystem health. Five artificial materials commonly used for biofilm growth (glass, stainless steel, aluminum, polypropylene, polyethylene) were tested to determine the one giving most robust and reproducible results. The effect of used sampler material on total microbial composition was not statistically significant; however, the non-plastic materials (glass, metal) gave more stable outputs without irregularities among sample parallels. The bias of the method is assessed with respect to the employment of a non-quantitative step (PCR amplification) to obtain quantitative results (relative abundance of identified taxa). This aspect is often overlooked in ecological and medical studies. We document that sequencing of a mixture of three merged primary PCR reactions for each sample and further evaluation of median values from three technical replicates for each sample enables to overcome this bias and gives robust and repeatable results well distinguishing among sampling localities and seasons.

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

Natural pyrrolobenzodiazepines (PBDs) form a large and structurally diverse group of antitumour microbial metabolites produced through complex pathways, which are encoded within biosynthetic gene clusters. We sequenced the gene cluster of limazepines and proposed their biosynthetic pathway based on comparison with five available gene clusters for the biosynthesis of other PBDs. Furthermore, we tested two recombinant proteins from limazepine biosynthesis, Lim5 and Lim6, with the expected substrates in vitro. The reactions monitored by LC-MS revealed that limazepine biosynthesis involves a new way of 3-hydroxyanthranilic acid formation, which we refer to as the chorismate/DHHA pathway and which represents an alternative to the kynurenine pathway employed for the formation of the same precursor in the biosynthesis of other PBDs. The chorismate/DHHA pathway is presumably also involved in the biosynthesis of PBD tilivalline, several natural products unrelated to PBDs, and its part is shared also with phenazine biosynthesis. The similarities between limazepine and phenazine biosynthesis indicate tight evolutionary links between these groups of compounds.

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

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.

Effects of Palladium(II) Chlorocomplex Speciation on the Controlled Interaction with a Polyaniline Film in Acid.

In this study, we determined the Pd(II) chlorocomplex species that has the most favorable interaction with an electropolymerized and protonated polyaniline (PANI) film. This study was completed with the intent to use this species to electrochemically build atomic palladium clusters in the PANI matrix. Varying amounts of NaCl were added to a K2PdCl4/HClO4 solution to result in three species studied: PdCl2(H2O)2, PdCl3(H2O)-, and PdCl42-. UV-vis spectroscopy was used to confirm the speciation, and Raman spectroscopy, X-ray photoelectron spectroscopy, and cyclic voltammograms were used to probe the interaction between the Pd species and PANI. It was determined that PdCl3(H2O)- most effectively interacts with PANI as a result of the charge balance between the anion and the protonated nitrogen-containing groups in the polymer. It has been also found that some fraction of inserted Pd(II) cannot be reduced to Pd(0).

Diversity of Alkylproline Moieties in Pyrrolobenzodiazepines Arises from Postcondensation Modifications of a Unified Building Block.

Anticancer pyrrolobenzodiazepines (PBDs) are one of several groups of natural products that contain unusual 4-alkyl-l-proline derivatives (APDs) in their structure. APD moieties of PBDs are characterized by high structural diversity achieved through unknown biosynthetic machinery. Based on LC-MS analysis of culture broths, feeding experiments, and protein assays, we show that APDs are not incorporated into PBDs in their final form as was previously hypothesized. Instead, a uniform building block, 4-propylidene-l-proline or 4-ethylidene-l-proline, enters the condensation reaction. The subsequent postcondensation steps are initiated by the introduction of an additional double bond catalyzed by a FAD-dependent oxidoreductase, which we demonstrated with Orf7 from anthramycin biosynthesis. The resulting double bond arrangement presumably represents a prerequisite for further modifications of the APD moieties. Our study gives general insight into the diversification of APD moieties of natural PBDs and provides proof-of-principle for precursor directed and combinatorial biosynthesis of new PBD-based antitumor compounds.

Lewis Acid Based Sorption of Trace Amounts of RuCl3 by Polyaniline.

A sorption process of RuCl3 in phosphate buffer by polyaniline (PANI) powder chemically synthesized from phosphoric acid was spectrophotometrically monitored as a function of time. It was determined that the sorption process follows the Langmuir and Freundlich isotherms, and their constants were evaluated. It was determined that chemisorption was the rate-controlling step. By conducting detailed studies, we assigned the chemisorption to Lewis acid based interactions of the sorbent electron pair localized at the benzenoid amine (-NH2) and quinoid imine (═NH) groups, with the sorbate, RuCl3, as the electron acceptor. The stability of the interaction over a period of ∼1 week showed that the presence of the Ru(III) in the PANI matrix reverses its state from emeraldine base to emeraldine salt, resulting in a change of conductivity. The partial electron donor based charge transfer is a slow process as compared to the sorption process involving Brønsted acid doping.

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?

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.

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

The immediate post-condensation steps in lincomycin biosynthesis are reminiscent of the mycothiol-dependent detoxification system of actinomycetes. This machinery provides the last proven lincomycin intermediate, a mercapturic acid derivative, which formally represents the 'waste product' of the detoxification process. We identified and purified new lincomycin intermediates from the culture broth of deletion mutant strains of Streptomyces lincolnensis and tested these compounds as substrates for proteins putatively involved in lincomycin biosynthesis. The results, based on LC-MS, in-source collision-induced dissociation mass spectrometry and NMR analysis, revealed the final steps of lincomycin biosynthesis, i.e. conversion of the mercapturic acid derivative to lincomycin. Most importantly, we show that deacetylation of the N'-acetyl-S-cysteine residue of the mercapturic acid derivative is required to 'escape' the detoxification-like system and proceed towards completion of the biosynthetic pathway. Additionally, our results, supported by l-cysteine-13C3, 15N incorporation experiments, give evidence that a different type of reaction catalysed by the homologous pair of pyridoxal-5'-phosphate-dependent enzymes, LmbF and CcbF, forms the branch point in the biosynthesis of lincomycin and celesticetin, two related lincosamides.

ClpP-independent function of ClpX interferes with telithromycin resistance conferred by Msr(A) in Staphylococcus aureus.

The ABCF family protein Msr(A) confers high resistance to macrolides but only low resistance to ketolides in staphylococci. Mutations in conserved functional regions of ClpX as well as deletion of clpX significantly increased Msr(A)-mediated resistance to the ketolide antibiotic telithromycin. ClpX is the chaperone component of the ClpXP two-component proteolytic system. Nevertheless, no changes in resistance were observed in a clpP knockout strain expressing msr(A), demonstrating that ClpX affects Msr(A) independently of ClpP.

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

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.