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1.
Science ; 383(6684): 721-726, 2024 Feb 16.
Article in English | MEDLINE | ID: mdl-38359125

ABSTRACT

We report the design conception, chemical synthesis, and microbiological evaluation of the bridged macrobicyclic antibiotic cresomycin (CRM), which overcomes evolutionarily diverse forms of antimicrobial resistance that render modern antibiotics ineffective. CRM exhibits in vitro and in vivo efficacy against both Gram-positive and Gram-negative bacteria, including multidrug-resistant strains of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. We show that CRM is highly preorganized for ribosomal binding by determining its density functional theory-calculated, solution-state, solid-state, and (wild-type) ribosome-bound structures, which all align identically within the macrobicyclic subunits. Lastly, we report two additional x-ray crystal structures of CRM in complex with bacterial ribosomes separately modified by the ribosomal RNA methylases, chloramphenicol-florfenicol resistance (Cfr) and erythromycin-resistance ribosomal RNA methylase (Erm), revealing concessive adjustments by the target and antibiotic that permit CRM to maintain binding where other antibiotics fail.


Subject(s)
Anti-Bacterial Agents , Bridged-Ring Compounds , Drug Resistance, Multiple, Bacterial , Lincosamides , Oxepins , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Erythromycin/chemistry , Erythromycin/pharmacology , Microbial Sensitivity Tests , Staphylococcus aureus/drug effects , Escherichia coli/drug effects , Pseudomonas aeruginosa/drug effects , Bridged-Ring Compounds/chemical synthesis , Bridged-Ring Compounds/chemistry , Bridged-Ring Compounds/pharmacology , Oxepins/chemical synthesis , Oxepins/chemistry , Oxepins/pharmacology , Lincosamides/chemical synthesis , Lincosamides/chemistry , Lincosamides/pharmacology , Animals , Mice , Drug Design , Ribosomes/chemistry
2.
Chembiochem ; 25(6): e202300840, 2024 03 15.
Article in English | MEDLINE | ID: mdl-38165257

ABSTRACT

Lincosamides are naturally occurring antibiotics isolated from Streptomyces sp. Currently, lincomycin A and its semisynthetic analogue clindamycin are used as clinical drugs. Due to their unique structures and remarkable biological activities, derivatizations of lincosamides via semi-synthesis and biosynthetic studies have been reported. This review summarizes the structures and biological activities of lincosamides, and the recent studies of lincosamide biosynthetic enzymes.


Subject(s)
Anti-Bacterial Agents , Lincomycin , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Lincosamides/pharmacology , Lincosamides/chemistry , Lincomycin/chemistry , Macrolides
3.
Angew Chem Int Ed Engl ; 62(29): e202304989, 2023 07 17.
Article in English | MEDLINE | ID: mdl-37222528

ABSTRACT

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.


Subject(s)
Anti-Bacterial Agents , Lincomycin , Glycosylation , Anti-Bacterial Agents/chemistry , Lincosamides/chemistry , Lincosamides/metabolism , Lincomycin/chemistry , Glycosyltransferases/metabolism , Crystallography, X-Ray
4.
J Am Chem Soc ; 143(18): 6829-6835, 2021 05 12.
Article in English | MEDLINE | ID: mdl-33930268

ABSTRACT

The development of a flexible, component-based synthetic route to the amino sugar fragment of the lincosamide antibiotics is described. This route hinges on the application and extension of nitroaldol chemistry to forge strategic bonds within complex amino sugar targets and employs a glycal epoxide as a versatile glycosyl donor for the installation of anomeric groups. Through building-block exchange and late-stage functionalization, this route affords access to a host of rationally designed lincosamides otherwise inaccessible by semisynthesis and underpins a platform for the discovery of new lincosamide antibiotics.


Subject(s)
Anti-Bacterial Agents/chemical synthesis , Lincosamides/chemical synthesis , Anti-Bacterial Agents/chemistry , Lincosamides/chemistry , Molecular Conformation
5.
Proc Natl Acad Sci U S A ; 117(40): 24794-24801, 2020 10 06.
Article in English | MEDLINE | ID: mdl-32958639

ABSTRACT

The structure of lincomycin A consists of the unusual eight-carbon thiosugar core methyllincosamide (MTL) decorated with a pendent N-methylprolinyl moiety. Previous studies on MTL biosynthesis have suggested GDP-ᴅ-erythro-α-ᴅ-gluco-octose and GDP-ᴅ-α-ᴅ-lincosamide as key intermediates in the pathway. However, the enzyme-catalyzed reactions resulting in the conversion of GDP-ᴅ-erythro-α-ᴅ-gluco-octose to GDP-ᴅ-α-ᴅ-lincosamide have not yet been elucidated. Herein, a biosynthetic subpathway involving the activities of four enzymes-LmbM, LmbL, CcbZ, and CcbS (the LmbZ and LmbS equivalents in the closely related celesticetin pathway)-is reported. These enzymes catalyze the previously unknown biosynthetic steps including 6-epimerization, 6,8-dehydration, 4-epimerization, and 6-transamination that convert GDP-ᴅ-erythro-α-ᴅ-gluco-octose to GDP-ᴅ-α-ᴅ-lincosamide. Identification of these reactions completes the description of the entire lincomycin biosynthetic pathway. This work is significant since it not only resolves the missing link in octose core assembly of a thiosugar-containing natural product but also showcases the sophistication in catalytic logic of enzymes involved in carbohydrate transformations.


Subject(s)
Lincomycin/biosynthesis , Streptomyces/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biosynthetic Pathways , Lincomycin/chemistry , Lincosamides/chemistry , Lincosamides/metabolism , Streptomyces/chemistry , Streptomyces/enzymology , Streptomyces/genetics
6.
Acc Chem Res ; 51(6): 1496-1506, 2018 06 19.
Article in English | MEDLINE | ID: mdl-29792672

ABSTRACT

Natural products typically are small molecules produced by living organisms. These products possess a wide variety of biological activities and thus have historically played a critical role in medicinal chemistry and chemical biology either as chemotherapeutic agents or as useful tools. Natural products are not synthesized for use by human beings; rather, living organisms produce them in response to various biochemical processes and environmental concerns, both internal and external. These processes/concerns are often dynamic and thus motivate the diversification, optimization, and selection of small molecules in line with changes in biological function. Consequently, the interactions between living organisms and their environments serve as an engine that drives coevolution of natural products and their biological functions and ultimately programs the constant theme of small-molecule development in nature based on biosynthesis generality and specificity. Following this theme, we herein review the biosynthesis of lincosamide antibiotics and dissect the process through which nature creates an unusual eight-carbon aminosugar (lincosamide) and then functionalizes this common high-carbon chain-containing sugar core with diverse l-proline derivatives and sulfur appendages to form individual members, including the clinically useful anti-infective agent lincomycin A and its naturally occurring analogues celesticetin and Bu-2545. The biosynthesis of lincosamide antibiotics is unique in that it results from an intersection of anabolic and catabolic chemistry. Many reactions that are usually involved in degradation and detoxification play a constructive role in biosynthetic processes. Formation of the trans-4-propyl-l-proline residue in lincomycin A biosynthesis requires an oxidation-associated degradation-like pathway composed of heme peroxidase-catalyzed ortho-hydroxylation and non-heme 2,3-dioxygenase-catalyzed extradiol cleavage for l-tyrosine processing prior to the building-up process. Mycothiol (MSH) and ergothioneine (EGT), two small-molecule thiols that are known for their redox-relevant roles in protection against various endogenous and exogenous stresses, function through two unusual S-glycosylations to mediate an eight-carbon aminosugar transfer, activation, and modification during the molecular assembly and tailoring processes in lincosamide antibiotic biosynthesis. Related intermediates include an MSH S-conjugate, mercapturic acid, and a thiomethyl product, which are reminiscent of intermediates found in thiol-mediated detoxification metabolism. In these biosynthetic pathways, "old" protein folds can result in "new" enzymatic activity, such as the DinB-2 fold protein for thiol exchange between EGT and MSH, the γ-glutamyltranspeptidase homologue for C-C bond cleavage, and the pyridoxal-5'-phosphate-dependent enzyme for diverse S-functionalization, generating interest in how nature develops remarkably diverse biochemical functions using a limited range of protein scaffolds. These findings highlight what we can learn from natural product biosynthesis, the recognition of its generality and specificity, and the natural theme of the development of bioactive small molecules, which enables the diversification process to advance and expand small-molecule functions.


Subject(s)
Anti-Bacterial Agents/biosynthesis , Lincosamides/biosynthesis , Anti-Bacterial Agents/chemistry , Glycosylation , Hydroxylation , Lincosamides/chemistry , Lincosamides/genetics , Multigene Family , Oxidation-Reduction
7.
Eur J Med Chem ; 146: 60-67, 2018 Feb 25.
Article in English | MEDLINE | ID: mdl-29396363

ABSTRACT

In erythromycin-resistant bacteria, the N6 position of A2058 in 23S rRNA is mono- or dimethylated by Erm family methyltransferases. This modification results in cross-resistance to macrolides, lincosamides and streptogramin B. Most inhibitors of Erm methyltransferases developed up-to-date target the cofactor-binding pocket, resulting in a lack of selectivity whereas inhibitors that bind the substrate-binding pocket demonstrate low in vitro activity. In this study, a molecular docking approach followed by biochemical screening was applied to search for inhibitors targeting both cofactor- and substrate-binding pockets of ErmC' methyltransferase. Based on the results of the molecular docking-based virtual screening of the clean-leads subset of the ZINC database, 29 compounds were chosen for experimental verification. Among them inhibitor 28 (ZINC code 32747906), with an IC50 of 100 µM, decreased the minimal inhibitory concentration of erythromycin in the Escherichia coli strain overexpressing ErmC'. Docking analysis of 28 to the ErmC' structure and the competitive ligand binding assay revealed a non-competitive model of inhibition. Inhibitor 28 served as a template for similarity-based virtual screening, which resulted in the identification of two derivatives 3s (ZINC code 62022572) and 4s (ZINC code 49032257) with an IC50 of 116 µM and 110 µM, respectively. Our results provide a basis for the development of inhibitors against the Erm-family of enzymes.


Subject(s)
Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial/drug effects , Enzyme Inhibitors/pharmacology , Escherichia coli/drug effects , Lincosamides/pharmacology , Macrolides/pharmacology , Methyltransferases/antagonists & inhibitors , Streptogramin Group B/pharmacology , Anti-Bacterial Agents/chemistry , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Lincosamides/chemistry , Macrolides/chemistry , Methyltransferases/metabolism , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , Streptogramin Group B/chemistry , Structure-Activity Relationship
8.
Nucleic Acids Res ; 45(17): 10284-10292, 2017 Sep 29.
Article in English | MEDLINE | ID: mdl-28973455

ABSTRACT

Antimicrobial resistance within a wide range of pathogenic bacteria is an increasingly serious threat to global public health. Among these pathogenic bacteria are the highly resistant, versatile and possibly aggressive bacteria, Staphylococcus aureus. Lincosamide antibiotics were proved to be effective against this pathogen. This small, albeit important group of antibiotics is mostly active against Gram-positive bacteria, but also used against selected Gram-negative anaerobes and protozoa. S. aureus resistance to lincosamides can be acquired by modifications and/or mutations in the rRNA and rProteins. Here, we present the crystal structures of the large ribosomal subunit of S. aureus in complex with the lincosamides lincomycin and RB02, a novel semisynthetic derivative and discuss the biochemical aspects of the in vitro potency of various lincosamides. These results allow better understanding of the drugs selectivity as well as the importance of the various chemical moieties of the drug for binding and inhibition.


Subject(s)
Lincosamides/pharmacology , Ribosome Subunits, Large, Bacterial/drug effects , Staphylococcus aureus/drug effects , Benzamides/chemistry , Benzamides/pharmacology , Binding Sites , Clindamycin/chemistry , Clindamycin/pharmacology , Crystallization , Crystallography, X-Ray , Drug Resistance, Microbial , Galactosides/chemistry , Galactosides/pharmacology , Hydrogen Bonding , Lincomycin/chemistry , Lincomycin/pharmacology , Lincosamides/chemistry , Molecular Structure , Ribosome Subunits, Large, Bacterial/ultrastructure , Staphylococcus aureus/ultrastructure , Static Electricity , Structure-Activity Relationship
9.
Int J Biol Macromol ; 103: 1-7, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28495631

ABSTRACT

We report the synthesis of Ag2S-Chitosan nanocomposites and Ag2S-chitosan nanohybrids as performance adsorbents for Lincosamides such as Clindamycin antibiotic removal. Isotherms and kinetic studies were determined to understand the adsorption behavior both two adsorbent. At low adsorbent dose, removals are increased in the adsorption process, and performance is better with Ag2S-chitosan nanohybrids due to the special surface area increased. The average sizes and surface area of Ag2S-Chitosan nanocomposites and Ag2S-chitosan nanohybrids were found as 50nm, 70nm and 180.18, 238.24m2g-1, respectively. In particular, Ag2S-Chitosan nanocomposites and Ag2S-chitosan nanohybrids show high maximum Clindamycin adsorption capacity (qmax) of 153.21, and 181.28mgg-1, respectively. More strikingly, Ag2S-Chitosan nanocomposites and Ag2S-chitosan nanohybrids are also demonstrated to nearly completely remove Clindamycin from drinking water. The excellent adsorption performance along with their cost effective, convenient synthesis makes this range of adsorbents highly promising for commercial applications in drinking water and wastewater treatment.


Subject(s)
Anti-Bacterial Agents/isolation & purification , Chitosan/chemistry , Lincosamides/isolation & purification , Nanocomposites/chemistry , Nanofibers/chemistry , Silver Compounds/chemical synthesis , Water Pollutants, Chemical/isolation & purification , Adsorption , Anti-Bacterial Agents/chemistry , Chemistry Techniques, Synthetic , Lincosamides/chemistry , Silver Compounds/chemistry , Wastewater/chemistry , Water Pollutants, Chemical/chemistry , Water Purification
10.
Biochem Pharmacol ; 133: 20-28, 2017 06 01.
Article in English | MEDLINE | ID: mdl-27940264

ABSTRACT

Lincomycin and its derivatives are antibiotics exhibiting biological activity against bacteria, especially Gram-positive ones, and also protozoans. Lincomycin and its semi-synthetic chlorinated derivative clindamycin are widely used in clinical practice. Both antibiotics are bacteriostatic, inhibiting protein synthesis in sensitive bacteria; however, at higher concentrations, they may be bactericidal. Clindamycin is usually much more active than lincomycin in the treatment of bacterial infections, in particular those caused by anaerobic species; it can also be used for the treatment of important protozoal diseases, e.g. malaria, most effectively in combination with other antibiotic or non-antibiotic antimicrobials (primaquine, fosfidomycin, benzoyl peroxide). Chemical structures of lincosamide antibiotics and the biosynthesis of lincomycin and its genetic control have been summarized and described. Resistance to lincomycin and clindamycin may be caused by methylation of 23S ribosomal RNA, modification of the antibiotics by specific enzymes or active efflux from the bacterial cell.


Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial/drug effects , Lincosamides/chemistry , Lincosamides/pharmacology , Animals , Anti-Bacterial Agents/therapeutic use , Bacterial Infections/diagnosis , Bacterial Infections/drug therapy , Drug Resistance, Bacterial/physiology , Humans , Lincosamides/therapeutic use , Structure-Activity Relationship
11.
Chembiochem ; 17(17): 1606-11, 2016 09 02.
Article in English | MEDLINE | ID: mdl-27431934

ABSTRACT

Lincosamides such as lincomycin A, celesticetin, and Bu-2545, constitute an important group of antibiotics. These natural products are characterized by a thiooctose linked to a l-proline residue, but they differ with regards to modifications of the thioacetal moiety, the pyrrolidine ring, and the octose core. Here we report that the pyridoxal 5'-phosphate-dependent enzyme CcbF (celesticetin biosynthetic pathway) is a decarboxylating deaminase that converts a cysteine S-conjugated intermediate into an aldehyde. In contrast, the homologous enzyme LmbF (lincomycin biosynthetic pathway) catalyzes C-S bond cleavage of the same intermediate to afford a thioglycoside. We show that Ccb4 and LmbG (downstream methyltransferases) convert the aldehyde and thiol intermediates into a variety of methylated lincosamide compounds including Bu-2545. The substrates used in these studies are the ß-anomers of the natural substrates. The findings not only provide insight into how the biosynthetic pathway of lincosamide antibiotics can bifurcate to generate different lincosamides, but also reveal the promiscuity of the enzymes involved.


Subject(s)
Biocatalysis , Cysteine/metabolism , Lincosamides/biosynthesis , Methyltransferases/metabolism , Biosynthetic Pathways , Cysteine/chemistry , Lincosamides/chemistry , Molecular Structure , Streptomyces/enzymology
12.
J Am Chem Soc ; 138(20): 6348-51, 2016 05 25.
Article in English | MEDLINE | ID: mdl-27171737

ABSTRACT

Pyridoxal-5'-phosphate (PLP)-dependent proteins constitute one of the largest and most important families of enzymes in living organisms. These proteins participate in numerous biochemical processes, many of which have not been characterized, and transform substrates containing an amino group through various reactions that share aldimine as a common intermediate. Herein, we report that the PLP-dependent enzymes CcbF and LmbF, which are highly related in phylogenesis, process cysteine S-conjugated intermediates in different ways and associate with individual downstream enzyme(s) toward distinct S-functionalization of the lincosamide antibiotics celesticetin and lincomycin A. CcbF catalyzes an unusual conversion that involves decarboxylation-coupled oxidative deamination of the cysteinyl group during the formation of a two-carbon alcohol linker, whereas LmbF is responsible for ß-elimination, followed by S-methylation to produce a methylmercapto group. The two tailoring routes are variable and exchangeable with each other, allowing for in vitro combinatorial biosynthesis of a number of hybrid lincosamide antibiotics, including the natural product Bu-2545. These findings demonstrate the wide diversity of PLP chemistry in enzymatic catalysis and its promising applicability in creation of new molecules.


Subject(s)
Anti-Bacterial Agents/chemistry , Cysteine/chemistry , Lincosamides/chemistry , Pyridoxal Phosphate/chemistry , Catalysis
13.
J Agric Food Chem ; 64(12): 2635-40, 2016 Mar 30.
Article in English | MEDLINE | ID: mdl-26971558

ABSTRACT

Calf milk replacers are low-cost feeds that contain available, digestible protein. During their reconstitution, however, the addition of drugs, such as antibiotics, could make them a very simple route for illicit treatment for therapeutic, preventive, or growth-promoting purposes. We developed an HPLC-MS/MS method, preceded by a unique extraction step, able to identify 17 antibiotics from seven classes (penicillins, tetracyclines, fluoroquinolones, sulfonamides, cephalosporins, amphenicols, and lincosamides) in this matrix. Prior to solid phase extraction (SPE), the sample underwent deproteinization and defatting. The method was fully validated according to Commission Decision 2002/657/EC. Decision limits (CCα) and detection capability (CCß) were in the ranges of 0.13-1.26 and 0.15-1.47 ng/mL, respectively. Thirty-eight samples were finally analyzed, showing the occasional presence of marbofloxacin (six samples) and amoxicillin (one sample).


Subject(s)
Anti-Infective Agents/analysis , Chromatography, Liquid/methods , Food, Formulated/microbiology , Milk/microbiology , Tandem Mass Spectrometry/methods , Amoxicillin/analysis , Amoxicillin/chemistry , Animals , Anti-Infective Agents/chemistry , Cattle , Cephalosporins/analysis , Cephalosporins/chemistry , Chromatography, High Pressure Liquid , Fluoroquinolones/analysis , Fluoroquinolones/chemistry , Lincosamides/analysis , Lincosamides/chemistry , Molecular Structure , Penicillins/analysis , Penicillins/chemistry , Sulfonamides/analysis , Sulfonamides/chemistry , Tetracyclines/analysis , Tetracyclines/chemistry
14.
J Mol Biol ; 427(12): 2229-43, 2015 Jun 19.
Article in English | MEDLINE | ID: mdl-25900373

ABSTRACT

One of the main mechanisms of resistance to lincosamide and aminoglycoside antibiotics is their inactivation by O-nucleotidylyltransferases (NTases). Significant sequence variation of lincomycin nucleotidylyltransferase (Lnu) and aminoglycoside nucleotidylyltransferase (ANT) enzymes plus lack of detailed information about the molecular basis for specificity of these enzymes toward chemically distinct antibiotic scaffolds hinders development of a general strategy to curb this resistance mechanism. We conducted an extensive sequence analysis identifying 129 putative antibiotic NTases constituting six distinct subfamilies represented by Lnu(A), Lnu(B), Lnu(C), Lnu(D), Lnu(F)/(G) plus ANT(2") enzymes. Since only the Lnu(B) enzyme has been previously studied in detail, we biochemically characterized the Lnu(A) and Lnu(D) enzymes, with the former representing the most sequence distinct Lnu ortholog. We also determined the crystal structure of the Lnu(A) enzyme in complex with a lincosamide. These data suggested that, while sharing the N-terminal nucleotidylyltransferase domain, the groups of antibiotic NTases feature structurally distinct C-terminal domains (CTDs) adapted to accommodate antibiotics. Comparative structural analysis among antibiotic NTases rationalized their specificity toward lincosamides versus aminoglycosides through active-site plasticity, which allows retention of general catalytic activity while accepting alterations at multiple, specific positions contributed by both domains. Based on this structural analysis, we suggest that antibiotic NTases evolved from an ancestral nucleotidylyltransferase along independent paths according to the identified groups, characterized by structural changes in the active site and recruitment of structurally diverse CTDs. These data show the complexity of enzyme-driven antibiotic resistance and provide a basis for broadly active inhibitors by identifying the key unifying features of antibiotic NTases.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Drug Resistance, Bacterial , Nucleotidyltransferases/chemistry , Nucleotidyltransferases/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Catalytic Domain , Cluster Analysis , Crystallography, X-Ray , Lincosamides/chemistry , Lincosamides/metabolism , Molecular Sequence Data , Nucleotidyltransferases/genetics , Phylogeny , Protein Binding , Protein Conformation , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity
15.
J Photochem Photobiol B ; 138: 324-30, 2014 Sep 05.
Article in English | MEDLINE | ID: mdl-25033467

ABSTRACT

The interaction of clindamycin phosphate (CP) with bovine serum albumin (BSA) is studied by using fluorescence spectra, UV-visible absorption, synchronous fluorescence spectra (SFS), CD, 3D fluorescence spectra and lifetime measurements under simulated physiological conditions. CP effectively quenched intrinsic fluorescence of BSA. The binding constants KA values are 2.540×10(5), 4.960×10(5), 7.207×10(5) L mol(-1), the number of binding sites n and corresponding thermodynamic parameters ΔG(o), ΔH(o) and ΔS(o) between CP and BSA were calculated at different temperatures. The interaction between CP and BSA occurs through dynamic quenching and the effect of CP on the conformation of BSA was also analyzed using SFS. The average binding distance r between the donor (BSA) and acceptor (CP) was determined based on Förster's theory. The results of fluorescence spectra, UV-vis absorption spectra and SFS show that the secondary structure of the protein has been changed in the presence of CP.


Subject(s)
Anti-Bacterial Agents/chemistry , Clindamycin/analogs & derivatives , Metals/chemistry , Serum Albumin, Bovine/chemistry , Animals , Anti-Bacterial Agents/metabolism , Binding Sites , Cattle , Clindamycin/chemistry , Clindamycin/metabolism , Energy Transfer , Ions/chemistry , Kinetics , Lincosamides/chemistry , Lincosamides/metabolism , Metals/metabolism , Protein Binding , Serum Albumin, Bovine/metabolism , Thermodynamics
16.
Chembiochem ; 14(17): 2259-62, 2013 Nov 25.
Article in English | MEDLINE | ID: mdl-24166757

ABSTRACT

Chemical diversity: Two SAM-dependent N-methyltransferases-LmbJ from the biosynthesis of the antibiotic lincomycin and CcbJ from celesticetin biosynthesis-have been characterized and compared. Both tested enzymes form multimers and are able to utilize N-demethyllincomycin, the natural substrate of LmbJ, with comparable efficiency.


Subject(s)
Anti-Bacterial Agents/biosynthesis , Biocatalysis , Lincomycin/biosynthesis , Lincosamides/biosynthesis , Methyltransferases/metabolism , Anti-Bacterial Agents/chemistry , Lincomycin/chemistry , Lincosamides/chemistry , Methyltransferases/chemistry , Molecular Conformation , Substrate Specificity
17.
PLoS One ; 8(12): e84902, 2013.
Article in English | MEDLINE | ID: mdl-24386435

ABSTRACT

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.


Subject(s)
Bacterial Proteins/chemistry , Dipeptides/chemistry , Evolution, Molecular , Lincomycin/biosynthesis , Lincosamides/biosynthesis , Molecular Dynamics Simulation , Streptomyces/enzymology , Lincomycin/chemistry , Lincosamides/chemistry , Protein Structure, Tertiary
18.
Antimicrob Agents Chemother ; 56(9): 4746-52, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22733067

ABSTRACT

The search for a specific rRNA methylase motif led to the identification of the new macrolide, lincosamide, and streptogramin B resistance gene erm(43) in Staphylococcus lentus. An inducible resistance phenotype was demonstrated by cloning and expressing erm(43) and its regulatory region in Staphylococcus aureus. The erm(43) gene was detected in two different DNA fragments, of 6,230 bp and 1,559 bp, that were each integrated at the same location in the chromosome in several S. lentus isolates of human, dog, and chicken origin.


Subject(s)
Adhesins, Bacterial/genetics , Chromosomes, Bacterial , DNA, Bacterial , Methyltransferases/genetics , Staphylococcus/genetics , Adhesins, Bacterial/chemistry , Adhesins, Bacterial/classification , Adhesins, Bacterial/metabolism , Amino Acid Sequence , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Chickens , Cloning, Molecular , Dogs , Drug Resistance, Bacterial , Humans , Lincosamides/chemistry , Lincosamides/pharmacology , Macrolides/chemistry , Macrolides/pharmacology , Methyltransferases/chemistry , Methyltransferases/classification , Methyltransferases/metabolism , Molecular Sequence Data , Phylogeny , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Staphylococcal Infections/drug therapy , Staphylococcal Infections/microbiology , Staphylococcus/classification , Staphylococcus/enzymology , Staphylococcus aureus/enzymology , Staphylococcus aureus/genetics , Streptogramin B/chemistry , Streptogramin B/pharmacology
19.
J Mol Model ; 18(6): 2727-40, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22116607

ABSTRACT

Lincosamides are a class of antibiotics used both in clinical and veterinary practice for a wide range of pathogens. This group of drugs inhibits the activity of the bacterial ribosome by binding to the 23S RNA of the large ribosomal subunit and blocking protein synthesis. Currently, three X-ray structures of the ribosome in complex with clindamycin are available in the Protein Data Bank, which reveal that there are two distinct conformations of the pyrrolidinyl propyl group of the bound clindamycin. In this work, we used quantum mechanical methods to investigate the probable conformations of clindamycin in order to explain the two binding modes in the ribosomal 23S RNA. We studied three lincosamide antibiotics: clindamycin, lincomycin, and pirlimycin at the B3LYP level with the 6-31G** basis set. The focus of our work was to connect the conformational landscape and electron densities of the two clindamycin conformers found experimentally with their physicochemical properties. For both functional conformers, we applied natural bond orbital (NBO) analysis and the atoms in molecules (AIM) theory, and calculated the NMR parameters. Based on the results obtained, we were able to show that the structure with the intramolecular hydrogen bond C=O…H-O is the most stable conformer of clindamycin. The charge transfer between the pyrrolidine-derivative ring and the six-atom sugar (methylthiolincosamide), which are linked via an amide bond, was found to be the dominant factor influencing the high stability of this conformer.


Subject(s)
Anti-Bacterial Agents/chemistry , Computer Simulation , Lincosamides/chemistry , Models, Molecular , Quantum Theory , Hydrogen Bonding , Molecular Conformation , Thermodynamics
20.
J Antimicrob Chemother ; 66 Suppl 6: vi37-45, 2011 Dec.
Article in English | MEDLINE | ID: mdl-22096065

ABSTRACT

BACKGROUND: Data on more than a decade of outpatient macrolide, lincosamide and streptogramin (MLS) use in Europe were collected from 33 countries within the European Surveillance of Antimicrobial Consumption (ESAC) project, funded by the European Centre for Disease Prevention and Control (ECDC), using the WHO Anatomical Therapeutic Chemical (ATC)/defined daily dose (DDD) methodology. METHODS: For the period 1997-2009, data on outpatient use of systemic MLS aggregated at the level of the active substance were collected and expressed in DDD (WHO, version 2011) per 1000 inhabitants per day (DID). Using a classification based on mean plasma elimination half-life, macrolide use was analysed for trends over time, seasonal variation and composition. RESULTS: Total outpatient MLS use in 2009 varied by a factor of 18 between the countries with highest (11.5 DID in Greece) and lowest (0.6 DID in Sweden) use. MLS use showed high seasonal variation. Short-, intermediate- and long-acting macrolides were the most commonly used agents in 2, 25 and 5 countries, respectively (mainly erythromycin, clarithromycin and azithromycin, respectively). In Sweden, mainly lincosamides (clindamycin) were used. Lincosamide use was observed in all countries, while substantial use of a streptogramin was only seen in France (pristinamycin). For Europe, a significant increase in outpatient MLS use was found, as well as a significant seasonal variation, which increased over time from 1997 to 2009. Relative use of long-acting macrolides and lincosamides significantly increased over time with respect to intermediate-acting macrolides, and relative use of the latter increased with respect to short-acting macrolides. CONCLUSIONS: The observed differences between European countries in the levels of MLS use and the extreme seasonal variations in their use suggest that this subgroup of antibiotics is still prescribed inappropriately in many countries.


Subject(s)
Drug Utilization/statistics & numerical data , Lincosamides/therapeutic use , Macrolides/therapeutic use , Outpatients/statistics & numerical data , Streptogramins/therapeutic use , Ambulatory Care , Anti-Bacterial Agents/therapeutic use , Bacterial Infections/drug therapy , Drug Utilization/trends , Europe , Half-Life , Humans , Lincosamides/chemistry , Macrolides/chemistry , Pharmacoepidemiology/methods , Seasons , Statistics as Topic/methods
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