Here, we describe a fluorescent assay developed to study competitive binding of the glycopeptide antibiotics to live bacteria cells. This assay demonstrated that the mechanism of action of the lipoglycopeptide antibiotics strongly depends on the hydrophobicity of the substitutes, with the best antibacterial activity of the glycopeptide antibiotics equally sharing properties of binding to D-Ala-D-Ala residues of the nascent peptidoglycan and to the membrane.
- MeSH
- antibakteriální látky metabolismus MeSH
- barvení a značení MeSH
- buněčná stěna mikrobiologie MeSH
- Enterococcus faecium metabolismus MeSH
- enterokoky rezistentní vůči vankomycinu metabolismus MeSH
- fluorescence MeSH
- glykopeptidy metabolismus MeSH
- lipoglykopeptidy chemie metabolismus MeSH
- mikrobiální testy citlivosti MeSH
- peptidoglykan metabolismus MeSH
- rhodaminy chemie MeSH
- Staphylococcus aureus metabolismus MeSH
- teikoplanin analogy a deriváty chemie metabolismus MeSH
- vankomycin chemie metabolismus MeSH
- vazba proteinů fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- anthramycin chemie MeSH
- bakteriální proteiny chemie MeSH
- katalytická doména MeSH
- kyseliny fenylpyrohroznové chemie MeSH
- linkomycin chemie MeSH
- metylace MeSH
- molekulární struktura MeSH
- mutace MeSH
- prolin chemie MeSH
- Streptomyces chemie MeSH
- uhlík chemie MeSH
- vazba proteinů MeSH
- Publikační typ
- dopisy MeSH
- práce podpořená grantem MeSH
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.
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.
The effects in vitro of 2,3-dehydrosilybin and several galloyl esters and methyl ethers on the viability, proliferation, and migration of human umbilical vein endothelial cells (HUVECs) were evaluated. The monogalloyl esters were synthesized by a chemoselective esterification method or by Steglich esterification of suitably protected 2,3-dehydrosilybin (1) with protected gallic acid. 2,3-Dehydrosilybin (1) displayed more potent cytotoxic, antiproliferative, and antimigratory activities (IC50 12.0, 5.4, and 12.2 μM, respectively) than silybin. The methylated derivatives were less active, with the least potent being 3,7-di-O-methyl-2,3-dehydrosilybin (6). On the other hand, galloylation at C-7 OH and C-23 OH markedly increased the cytotoxicity and the effects on the proliferation and migration of HUVECs. The most active derivative was 7-O-galloyl-2,3-dehydrosilybin (13; IC50 value of 3.4, 1.6, and 4.7 μM in the cytotoxicity, inhibition of proliferation, and antimigratory assays, respectively). Overall, this preliminary structure-activity relationship study demonstrated the importance of a 2,3-double bond, a C-7 OH group, and a galloyl moiety in enhancing the activity of flavonolignans toward HUVECs.
- MeSH
- endoteliální buňky pupečníkové žíly (lidské) účinky léků MeSH
- kyselina gallová farmakologie MeSH
- lidé MeSH
- methylethery farmakologie MeSH
- molekulární struktura MeSH
- scavengery volných radikálů chemie MeSH
- silymarin chemie farmakologie MeSH
- viabilita buněk účinky léků MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- Publikační typ
- časopisecké články MeSH
A new, efficient, and general semisynthesis of hydnocarpin-type flavonolignans was developed and optimized, enabling gram-scale production of hydnocarpin D (2). Moreover, the syntheses of optically pure hydnocarpin isomers [(10R,11R)-hydnocarpin (1a), (10R,11R)-hydnocarpin D (2a), and (10S,11S)-hydnocarpin D (2b)], as well as the synthesis of isohydnocarpin (8), were achieved for the first time utilizing this new method. The synthesis is based on the two-step transformation of the readily available flavonolignans from milk thistle (Silybum marianum), accessible by isolation from the commercial extract silymarin. The first step relies on the regioselective formylation of the C-3 hydroxy group of the dihydroflavonol-type precursor using the Vilsmeier-Haack reagent, followed by formic acid elimination by triethylamine in the second step. The synthesized compounds were effective inhibitors of Staphylococcus aureus biofilm formation, with (10S,11S)-hydnocarpin D (2b) being the most potent inhibitor. Furthermore, the effect of glucose on biofilm formation was tested, and glucose decreased the biofilm inhibitory activity of 2b. Moreover, 2b increased the susceptibility of Staph. aureus to enrofloxacin.
- MeSH
- antioxidancia MeSH
- biofilmy účinky léků MeSH
- flavonolignany chemie izolace a purifikace farmakologie MeSH
- molekulární struktura MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- ostropestřec mariánský chemie MeSH
- silymarin chemie MeSH
- Staphylococcus aureus účinky léků MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
The biosynthetic gene cluster of porothramycin, a sequence-selective DNA alkylating compound, was identified in the genome of producing strain Streptomyces albus subsp. albus (ATCC 39897) and sequentially characterized. A 39.7 kb long DNA region contains 27 putative genes, 18 of them revealing high similarity with homologous genes from biosynthetic gene cluster of closely related pyrrolobenzodiazepine (PBD) compound anthramycin. However, considering the structures of both compounds, the number of differences in the gene composition of compared biosynthetic gene clusters was unexpectedly high, indicating participation of alternative enzymes in biosynthesis of both porothramycin precursors, anthranilate, and branched L-proline derivative. Based on the sequence analysis of putative NRPS modules Por20 and Por21, we suppose that in porothramycin biosynthesis, the methylation of anthranilate unit occurs prior to the condensation reaction, while modifications of branched proline derivative, oxidation, and dimethylation of the side chain occur on already condensed PBD core. Corresponding two specific methyltransferase encoding genes por26 and por25 were identified in the porothramycin gene cluster. Surprisingly, also methyltransferase gene por18 homologous to orf19 from anthramycin biosynthesis was detected in porothramycin gene cluster even though the appropriate biosynthetic step is missing, as suggested by ultra high-performance liquid chromatography-diode array detection-mass spectrometry (UHPLC-DAD-MS) analysis of the product in the S. albus culture broth.
- MeSH
- anthramycin analogy a deriváty biosyntéza chemie MeSH
- bakteriální proteiny genetika metabolismus MeSH
- molekulární sekvence - údaje MeSH
- molekulární struktura MeSH
- multigenová rodina * MeSH
- sekvenční analýza MeSH
- Streptomyces chemie genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
This paper describes an efficient oxime ligation strategy to prepare multivalent conjugates wherein peptides alone or in combination with carbohydrate or oxime groups were coupled to a cyclopeptide scaffold. To demonstrate the versatility of this approach, two classes of conjugates have been prepared. In one class, we attached two or four peptide sequences to the cyclopeptide core together with free oxime groups, while the second class contains an additional substitution with four or two monosaccharides. The well-defined structure of these conjugates was confirmed by high-resolution mass spectrometry.
