Biosynthesis of tylosin: Oxidations of 5-O-mycaminosylprtylonolide at C-20 and C-23 with a cell-free extract from Streptomycesfradiae

The enzymatic conversion of various tylosin precursors was carried out using a cell-free system of the tylosin producer $\text{Streptomyces}\text{fradiae}$ to determine the order and intermediates of oxidations of the 16-membered branched lactone ring at C-20 and C-23 in the biosynthesis of tylosin. It was found that the order of the oxidation of the lactone is: (1) hydroxylation of 5-$\text{O}$-mycaminosylprotylonolide at C-20, (2) oxidation of C-20 hydroxymethyl to formyl, (3) hydroxylation at C-23 to give 5-$\text{O}$-mycaminosyltylonolide. The formation of 23-hydroxy-5-$\text{O}$-mycaminosylprotylonolide from 5-$\text{O}$-mycaminosylprotylonolide was not observed.     

Properties of Ribosomes from Streptomyces erythreus and Streptomyces griseus

Ribosomes from an erythromycin-producing strain, Streptomyces erythreus, lacked affinity for erythromycin and were also resistant to other macrolide antibiotics (leucomycin, spiramycin, and tylosin) and to lincomycin, whereas Streptomyces griseus B(3) ribosomes were susceptible to all of these antibiotics.     

A defined system for hybrid macrolide biosynthesis in Saccharopolyspora erythraea

The biological activity of polyketide antibiotics is often strongly dependent on the presence and type of deoxysugar residues attached to the aglycone core. A system is described here, based on the erythromycin-producing strain of Saccharopolyspora erythraea, for detection of hybrid glycoside formation, and this system has been used to demonstrate that an amino sugar characteristic of 14-membered macrolides (D-desosamine) can be efficiently attached to a 16-membered aglycone substrate. First, the S. erythraea mutant strain DM was created by deletion of both eryBV and eryCIII genes encoding the respective ery glycosyltransferase genes. The glycosyltransferase OleG2 from Streptomyces antibioticus, which transfers L-oleandrose, has recently been shown to transfer rhamnose to the oxygen at C-3 of erythronolide B and 6-deoxyerythronolide B. In full accordance with this finding, when oleG2 was expressed in S. erythraea DM, 3-O-rhamnosyl-erythronolide B and 3-O-rhamnosyl-6-deoxyerythronolide B were produced. Having thus validated the expression system, endogenous aglycone production was prevented by deletion of the polyketide synthase (eryA) genes from S. erythraea DM, creating the triple mutant SGT2. To examine the ability of the mycaminosyltransferase TylM2 from Streptomyces fradiae to utilise a different amino sugar, tylM2 was integrated into S. erythraea SGT2, and the resulting strain was fed with the 16-membered aglycone tylactone, the normal TylM2 substrate. A new hybrid glycoside was isolated in good yield and characterized as 5-O-desosaminyl-tylactone, indicating that TylM2 may be a useful glycosyltransferase for combinatorial biosynthesis. 5-O-glucosyl-tylactone was also obtained, showing that endogenous activated sugars and glycosyltransferases compete for aglycone in these cells.     

Biosynthesis of bisorbicillinoid in Trichoderma sp. USF-2690; evidence for the biosynthetic pathway,via sorbicillinol,of sorbicillin,bisorbicillinol, bisorbibutenolide,and bisorbicillinolide

An incorporation study of [1-(13)C] and [1,2-(13)C2] labeled sodium acetates into sorbicillinol 1 established a ring closure system between C-1 and C-6 and the positions that were oxidized and/or methylated on a hexaketide chain. Subsequent investigations, using 13C-labeled 1 prepared from [1-(13)C] labeled sodium acetate, clearly demonstrated that both bisorbicillinol 2 and sorbicillin 6 incorporated 13C-labeled 1 into their carbon skeletons. 13C-labeled bisorbicillinols 2 derived from [1-(13)C]- and [2-(13)C]-labeled sodium acetates clearly indicate that these were on the biosynthetic route from 1 to bisorbibutenolide (bislongiquinolide) 3 and bisorbicillinolide 4 via 2 as a branching point in the fungus.     

Ribosomes from spiramycin resistant mutants of Escherichia coli Q13

Summary Mutants from Escherichia coli Q13 were selected for resistance to leucomycin, tylosin or spiramycin. Most of the mutants so selected exhibited cross resistance to all the macrolide antibiotics tested including erythromycin. A few mutants however seem to be less resistant to erythromycin. One mutant, QSP008, was highly resistant to tylosin, leucomycin and spiramycin but relatively sensitive to erythromycin. Another mutant, QSP006, was highly resistant to spiramycin but less resistant to erythromycin, tylosin and leucomycin. This selective resistance of cells to specific antibiotics could be due to the extent of conformational alteration of their ribosomes, which may be demonstrated by the extent of ¹⁴C-erythromycin binding to these ribosomes. The ribosomes from QSP008 cells were found to contain an altered 50-8 protein of the 50s ribosomal subunit, while in the ribosomes from QSP006 no such protein change could be detected by the methods used.A preliminary data of part of this work has been published (Tanaka, Teraoka, Tamaki, Watanabe, Osawa, Otaka, and Takata, 1971).     

Design and synthesis of novel leucomycin analogues modified at the C-3 position. Part II: 3-O-(3-Aryl-2-propenyl)leucomycin analogues

The design and synthesis of 16-membered macrolides modified at the C-3 position are described. Starting from fully protected intermediate (5), appropriate modifications including Heck reaction were performed to furnish 3-O-(3-aryl-2-propenyl)leucomycin A(7) analogues (9a-9m). These leucomycin A(7) derivatives showed improved in vitro antibacterial activities against clinically important pathogens including erythromycin-resistant Streptococcus pneumoniae (ERSP). SAR analysis of derivatives modified at the C-3 and C-3' positions suggested that single modification at C-3 or C-3' was effective for in vitro antibacterial activity.     

Biosynthesis of Bisorbicillinoid in Trichoderma sp. USF-2690; Evidence for the Biosynthetic Pathway,via Sorbicillinol,of Sorbicillin,Bisorbicillinol, Bisorbibutenolide,and…

An incorporation study of [1-¹³C] and [1,2-¹³C₂] labeled sodium acetates into sorbicillinol 1 established a ring closure system between C-1 and C-6 and the positions that were oxidized and/or methylated on a hexaketide chain. Subsequent investigations, using ¹³C-labeled 1 prepared from [1-¹³C] labeled sodium acetate, clearly demonstrated that both bisorbicillinol 2 and sorbicillin 6 incorporated ¹³C-labeled 1 into their carbon skeletons. ¹³C-labeled bisorbicillinols 2 derived from [1-¹³C]- and [2-¹³C]-labeled sodium acetates clearly indicate that these were on the biosynthetic route from 1 to bisorbibutenolide (bislongiquinolide) 3 and bisorbicillinolide 4 via 2 as a branching point in the fungus.     

Isolation of Cyclic 3′,5′-Guanosine Monophosphate from Bacterial Culture Fluids

To clarify the biosynthetic pathway of maridomycin, the biosynthetic relation among 16-membered macrolide antibiotics belonging to leucomycin-maridomycin group was examined with intact cells or growing cultures of Streptomyces hygroscopicus No. B–5050–HA and its mutants.

Carbomycin A, B and leucomycin A₃ were converted to maridomycin II in the pH range of 5.5-7.5 with an optimum between 6.0-6.5. But, maridomycin II was not converted to leucomycin A₃ or carbomycin B. These findings were confirmed by culture experiment. Leucomycin A₃, carbomycin A and B added to the culture at 48 hr were almost completely converted to maridomycin II.

On the basis of these facts, we propose a biosynthetic relation among leucomycin A₃, carbomycin A, carbomycin B and maridomycin II.     

Identification and characterization of the niddamycin polyketide synthase genes from Streptomyces caelestis.

The genes encoding the polyketide synthase (PKS) portion of the niddamycin biosynthetic pathway were isolated from a library of Streptomyces caelestis NRRL-2821 chromosomal DNA. Analysis of 40 kb of DNA revealed the presence of five large open reading frames (ORFs) encoding the seven modular sets of enzymatic activities required for the synthesis of a 16-membered lactone ring. The enzymatic motifs identified within each module were consistent with those predicted from the structure of niddamycin. Disruption of the second ORF of the PKS coding region eliminated niddamycin production, demonstrating that the cloned genes are involved in the biosynthesis of this compound.     

The structures of four macrolide antibiotics bound to the large ribosomal subunit

Crystal structures of the Haloarcula marismortui large ribosomal subunit complexed with the 16-membered macrolide antibiotics carbomycin A, spiramycin, and tylosin and a 15-membered macrolide, azithromycin, show that they bind in the polypeptide exit tunnel adjacent to the peptidyl transferase center. Their location suggests that they inhibit protein synthesis by blocking the egress of nascent polypeptides. The saccharide branch attached to C5 of the lactone rings extends toward the peptidyl transferase center, and the isobutyrate extension of the carbomycin A disaccharide overlaps the A-site. Unexpectedly, a reversible covalent bond forms between the ethylaldehyde substituent at the C6 position of the 16-membered macrolides and the N6 of A2103 (A2062, E. coli). Mutations in 23S rRNA that result in clinical resistance render the binding site less complementary to macrolides.     

Peptide Derivatives of Antibiotics Tylosin and Desmycosin, Protein Synthesis Inhibitors

Biologically active peptide derivatives of 16-member macrolide antibiotics were synthesized as potential probes for the investigation of nascent peptide chain topography in the ribosomal exit tunnel. The tylosin and desmycosin aldehyde groups at the C6 position of the lactone ring were modified by the aminooxyacetyl-L-alanyl-L-alanine methyl ester.     

Biosynthetic studies on saframycin A, a quinone antitumor antibiotic produced by Streptomyces lavendulae

The biosynthesis of saframycin A, a heterocyclic quinone antitumor antibiotic isolated from Streptomyces lavendulae 314, was studied by feeding experiments with 14C and 13C precursors. Highly increased production of saframycin A and prolongation of the maximum production period of saframycin A were attained by constant pH control of the culture and by addition of chloramphenicol to the culture. The biosynthetic origin of the quinone skeleton common to the saframycin group was confirmed to be two tyrosine molecules which condense to generate the basic ring system of saframycin A. Feeding experiments with [1-13C]tyrosine showed specific labeling of C-11 and C-21 carbons of saframycin A, and the enrichment of the carbons was 40-fold over natural abundance. Two O- and two C-methyl and one N-methyl carbons arose directly from methionine, and alanine and glycine were the precursors for the pyruvoyl amide side chain of saframycin A.     

Genetic architecture of the polyketide synthases for methymycin and pikromycin series macrolides

The methymycin and pikromycin series of antibiotics are structurally related macrolides produced by several Streptomyces species, including Streptomyces venezuelae ATCC 15439, which produces both 12-membered ring macrolides methymycin, neomethymycin, and 14-membered ring macrolides pikromycin and narbomycin. Cloning and sequencing of the biosynthetic gene clusters for these macrolides from three selected Streptomyces strains revealed a common genetic architecture of their polyketide synthases (PKSs). Unlike PKS clusters of other 14-membered ring macrolides such as erythromycin and oleandomycin, each of the pikromycin series producers harbors a six module PKS cluster, in which modules 5 and 6 are encoded on two separate proteins instead of one bimodular protein, as well as a thioesterase II gene immediately downstream of the main PKS gene. The results shed new light on the evolution of modular PKSs and provide further evidence on the regulation of methymycin and pikromycin production in S. venezuelae ATCC 15439.     

Biosynthesis of the macrolide antibiotic chlorothricin: basic building blocks

The biosynthesis of chlorothricin (I), a macrolide antibiotic isolated from Streptomyces antibioticus Tü 99, has been studied by feeding experiments with 14C- and 3H-labeled precursors. Acetate and propionate, but not methionine and mevalonate, were incorporated into the macrocylic aglycone of the antibiotic. Glucose and the various carbon atoms of tyrosine, except the carboxyl carbon, also contributed label to the aglycone. Glucose also seems to be a specific precursor of the 2-deoxyrhamnose moiety, probably via a process involving a hydrogen shift from C-4 to C-6 of the hexose. The substituted 6-methylsalicylic acid moiety seems to be derived from acetate and one O-methyl group provided by methionine; shikimic acid is not incorporated.     

Appearance in Japan of highly macrolide-resistant Escherichia coli producing macrolide 2'-phosphotransferase II.

Escherichia coli CU1, a clinical isolate recovered in Japan in 1997, was found to be highly-resistant to both 14-membered and 16-membered ring macrolide antibiotics. A crude extract prepared from strain CU1 inactivated 14-, 15- and 16-membered ring macrolides in the presence of ATP and the Rf value of inactivated oleandomycin was identical to that of oleandomycin 2'-phosphate. This suggested that strain CU1 produced the enzyme macrolide 2'-phosphotransferase [MPH(2')]. Substrate specificity of the crude enzyme from strain CU1 against 14-, 15- and 16-membered ring macrolides was basically similar to that of MPH(2')II from strain BM2506, differing in that the former more effectively inactivated roxithromycin and tylosin. Subsequent attempts were made to clone the novel mph gene encoding for MPH(2') in strain CU1. The mph gene carried by strain CU1 was located on nontransmissible plasmid DNA, designated pCU001. Its molecular weight, estimated by agarose electrophoresis, was approximately 57 kD. The DNA sequence of the cloned mph gene from the Japanese isolate CU1 was identical to that of mphB, which until now had only been recovered in France. The variance in the substrate specificity of MPH(2')II from each strain led us to speculate that other factors in the reaction affect the enzymatic inactivation activity.     

Biosynthesis of withanolides in Acnistus breviflorus

Administration of [2?¹⁴C]mevalonolactone to excised leaves of Acnistus breviflorus produced labelled withaferin A and jaborosalactone A. The former was degraded leading to the isolation of glyceric acid from C-25–C-27 of the withanolide. These carbons represented only 2 % of the total radioactivity of withaferin A. The relative radioactivity of these carbons indicated that C-26 is directly derived from C-2 of mevalonolactone suggesting that the 25-pro-R-methyl group of cholesterol or any other sterol intermediate had been oxidized to form the lactone ring of the withanolide. The total radioactivity value found for C-25–C-27 was much lower than the expected 20 % of the total value for the withanolide indicating that the side chain of the sterol precursor had been partially cleaved during the biosynthetic process.     

Stepwise binding of tylosin and erythromycin to Escherichia coli ribosomes, characterized by kinetic and footprinting analysis

Erythromycin and tylosin are 14- and 16-membered lactone ring macrolides, respectively. The current work shows by means of kinetic and chemical footprinting analysis that both antibiotics bind to Escherichia coli ribosomes in a two-step process. The first step established rapidly, involves a low-affinity binding site placed at the entrance of the exit tunnel in the large ribosomal subunit, where macrolides bind primarily through their hydrophobic portions. Subsequently, slow conformational changes mediated by the antibiotic hydrophilic portion push the drugs deeper into the tunnel, in a high-affinity site. Compared with erythromycin, tylosin shifts to the high-affinity site more rapidly, due to the interaction of the mycinose sugar of the drug with the loop of H35 in domain II of 23 S rRNA. Consistently, mutations of nucleosides U2609 and U754 implicated in the high-affinity site reduce the shift of tylosin to this site and destabilize, respectively, the final drug-ribosome complex. The weak interaction between tylosin and the ribosome is Mg2+ independent, unlike the tight binding. In contrast, both interactions between erythromycin and the ribosome are reduced by increasing concentrations of Mg2+ ions. Polyamines attenuate erythromycin affinity for the ribosome at both sequential steps of binding. In contrast, polyamines facilitate the initial binding of tylosin, but exert a detrimental, more pronounced, effect on the drug accommodation at its final position. Our results emphasize the role of the particular interactions that side chains of tylosin and erythromycin establish with 23 S rRNA, which govern the exact binding process of each drug and its response to the ionic environment.     

Inhibition of the ribosomal peptidyl transferase reaction by the mycarose moiety of the antibiotics carbomycin, spiramycin and tylosin

Many antibiotics, including the macrolides, inhibit protein synthesis by binding to ribosomes. Only some of the macrolides affect the peptidyl transferase reaction. The 16-member ring macrolide antibiotics carbomycin, spiramycin, and tylosin inhibit peptidyl transferase. All these have a disaccharide at position 5 in the lactone ring with a mycarose moiety. We have investigated the functional role of this mycarose moiety. The 14-member ring macrolide erythromycin and the 16-member ring macrolides desmycosin and chalcomycin do not inhibit the peptidyl transferase reaction. These drugs have a monosaccharide at position 5 in the lactone ring. The presence of mycarose was correlated with inhibition of peptidyl transferase, footprints on 23 S rRNA and whether the macrolide can compete with binding of hygromycin A to the ribosome. The binding sites of the macrolides to Escherichia coli ribosomes were investigated by chemical probing of domains II and V of 23 S rRNA. The common binding site is around position A2058, while effects on U2506 depend on the presence of the mycarose sugar. Also, protection at position A752 indicates that a mycinose moiety at position 14 in 16-member ring macrolides interact with hairpin 35 in domain II. Competitive footprinting of ribosomal binding of hygromycin A and macrolides showed that tylosin and spiramycin reduce the hygromycin A protections of nucleotides in 23 S rRNA and that carbomycin abolishes its binding. In contrast, the macrolides that do not inhibit the peptidyl transferase reaction bind to the ribosomes concurrently with hygromycin A. Data are presented to argue that a disaccharide at position 5 in the lactone ring of macrolides is essential for inhibition of peptide bond formation and that the mycarose moiety is placed near the conserved U2506 in the central loop region of domain V 23 S rRNA.     

Vitamin K dependent carboxylation: determination of the stereochemical course using 4-fluoroglutamyl-containing substrate

The stereochemical course of the vitamin K dependent carboxylation has been elucidated using a (4S)-4-fluoroglutamyl-containing pentapeptide as a substrate. The absolute configuration of the [13C]-4-carboxy-4-fluoroglutamate obtained when the carboxylation was carried out with 13C-labeled sodium bicarbonate, was determined after reduction of the [13C]-4-carboxy-4-fluoroglutamyl residue into 4-fluoro-5,5'-dihydroxyleucine, hydrolysis, lactonization, and peracetylation. The absolute configuration at C-4 was determined to be S by locating the 13C label in the lactone ring of the trans isomeric lactone and in the hydroxymethyl group of the cis isomer following HPLC separation of both isomers and analysis by GC/MS/MS techniques. It follows that the vitamin K dependent carboxylation occurs with inversion of configuration.