Modular polyketide synthase contains two reaction chambers that operate asynchronously.

Type I modular polyketide synthases are homodimeric multidomain assembly line enzymes that synthesize a variety of polyketide natural products by performing polyketide chain extension and ß-keto group modification reactions. We determined the 2.4-angstrom-resolution x-ray crystal structure and the 3.1-angstrom-resolution cryo­electron microscopy structure of the Lsd14 polyketide synthase, stalled at the transacylation and condensation steps, respectively. These structures revealed how the constituent domains are positioned relative to each other, how they rearrange depending on the step in the reaction cycle, and the specific interactions formed between the domains. Like the evolutionarily related mammalian fatty acid synthase, Lsd14 contains two reaction chambers, but only one chamber in Lsd14 has the full complement of catalytic domains, indicating that only one chamber produces the polyketide product at any given time.

Streptomyces lasalocidi sp. nov. (formerly 'Streptomyces lasaliensis'), an actinomycete isolated from soil which produces the polyether antibiotic lasalocid.

Strain ATCC 31180T was isolated from soil collected in Hyde Park, Massachusetts (USA), and found to produce the polyether antibiotic lasalocid. The name 'Streptomyces lasaliensis' has been in common use since 1974, without a recognized taxonomic description. The most closely related type cultures determined by rRNA gene sequence similarity were Streptomyces longwoodensis DSM 41677T (100 %) and Streptomyces galbus DSM 40089T (100 %). OrthoANI values with S. longwoodensis and S. galbus were 95.50 and 94.41 %, respectively. Chemotaxonomic characteristics supported inclusion within the genus Streptomyces. The cell wall peptidoglycan contained ll-diaminopimelic acid, and the major whole-cell sugars were glucose and ribose. Polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol, phosphatidylglycerol, one unidentified lipid and one unidentified glycolipid. The major menaquinones detected were MK9(H4), MK9(H6) and MK9(H8). The major cellular fatty acids were anteiso-C15 : 0, anteiso-C17 : 0, iso-C16 : 0, iso-C15 : 0 and anteiso-C17 : 1. Its DNA had a G+C content of 72.6 %. Differentiation of ATCC 31180T from the closely related species was evident from digital DNA-DNA hybridization values of 61.80 and 56.90 % for S. longwoodensis and S. galbus respectively. Significant differences were seen in the polyphasic phenotypic analyses. ATCC 31180T produced lasalocid, grew from 10 to 45 °C, pH4-8 and in the presence of 0-10 % NaCl, 0.01 % NaN3 and 1 % phenol. Melanin was produced; H2S and indole were not. Nitrate was not reduced. Spore chains were retinaculum-apertum and spore surfaces were smooth. Spore colour, mycelia colour and soluble pigment production were medium-dependent. The proposed name is Streptomyces lasalocidi sp. nov.; the type strain being ATCC 31180T (=NRRL 3382T=DSM 46487T).

Analysis of lasalocid residues in grease and fat using liquid chromatography-mass spectrometry.

A method for the determination of lasalocid, an antibiotic and coccidiostat, in grease and fat is described. The manufacture of lasalocid produces a grease-like residue as a waste byproduct. Recently this byproduct has been shown to have been illegally introduced into the animal feed chain. Therefore, a quantitative and confirmatory procedure to analyse for lasalocid in this matrix is needed. A portion of grease/oil sample was extracted into hexane-washed acetonitrile, and a portion of the extract was then applied to a carboxylic acid solid-phase extraction (SPE) column for concentration and clean-up. The SPE column was washed with additional hexane-washed acetonitrile and ethyl acetate/methanol, after which lasalocid was eluted with 10% ammoniated methanol. The eluate was evaporated to dryness, redissolved in (1:1) acetonitrile-water and filtered through a PTFE syringe filter. Confirmation and quantitation of lasalocid in the final extract employed a triple quadrupole LC-MS/MS. The method was applied to grease and oil samples containing from 0.02 to 34,000 mg kg(-1) of lasalocid.

Biosynthetic machinery of ionophore polyether lasalocid: enzymatic construction of polyether skeleton.

Diversity of natural polycyclic polyethers originated from very simple yet versatile strategy consisting of epoxidation of linear polyene followed by epoxide opening cascade. To understand two-step enzymatic transformations at molecular basis, a flavin containing monooxygenase (EPX) Lsd18 and an epoxide hydrolase (EH) Lsd19 were selected as model enzymes for extensive investigation on substrate specificity, catalytic mechanism, cofactor requirement and crystal structure. This pioneering study on prototypical lasalocid EPX and EH provides insight into detailed mechanism of ionophore polyether assembly machinery and clarified remaining issues for polyether biosynthesis.

Enzymatic catalysis of anti-Baldwin ring closure in polyether biosynthesis.

Polycyclic polyether natural products have fascinated chemists and biologists alike owing to their useful biological activity, highly complex structure and intriguing biosynthetic mechanisms. Following the original proposal for the polyepoxide origin of lasalocid and isolasalocid and the experimental determination of the origins of the oxygen and carbon atoms of both lasalocid and monensin, a unified stereochemical model for the biosynthesis of polyether ionophore antibiotics was proposed. The model was based on a cascade of nucleophilic ring closures of postulated polyepoxide substrates generated by stereospecific oxidation of all-trans polyene polyketide intermediates. Shortly thereafter, a related model was proposed for the biogenesis of marine ladder toxins, involving a series of nominally disfavoured anti-Baldwin, endo-tet epoxide-ring-opening reactions. Recently, we identified Lsd19 from the Streptomyces lasaliensis gene cluster as the epoxide hydrolase responsible for the epoxide-opening cyclization of bisepoxyprelasalocid A to form lasalocid A. Here we report the X-ray crystal structure of Lsd19 in complex with its substrate and product analogue to provide the first atomic structure-to our knowledge-of a natural enzyme capable of catalysing the disfavoured epoxide-opening cyclic ether formation. On the basis of our structural and computational studies, we propose a general mechanism for the enzymatic catalysis of polyether natural product biosynthesis.

Intriguing substrate tolerance of epoxide hydrolase Lsd19 involved in biosynthesis of the ionophore antibiotic lasalocid A.

Recently, we reported that the epoxide hydrolase Lsd19, the first enzyme shown to catalyze epoxide-opening cascades, can catalyze the conversion of a putative bisepoxide intermediate to polyether antibiotic lasalocid, which involves energetically disfavored 6-endo-tet cyclization of the epoxy alcohol. Here, we examined the substrate tolerance of Lsd19. Lsd19 accepts various substrate analogues differing in the left segment of lasalocid and epoxide stereochemistry to afford either THF-THP or THF-THF products with excellent regioselectivity.

Identification of a gene cluster of polyether antibiotic lasalocid from Streptomyces lasaliensis.

Elucidation of enzymatic polyether formation is a long-standing controversial issue in organic chemistry. To address this intriguing issue, identifying the actual substrate for epoxidation and sequential cyclization is essential. We selected the representative polyether ionophore, lasalocid, which has been proposed to undergo no modification at the late stage of biosynthesis. Cloning and a sequence analysis revealed seven polyketide synthase (PKS) genes, epoxidase and epoxide hydrolase genes for sequential ether formation, and several putative genes for supplying ethylmalonyl-CoA. Based on bioinformatic data, we propose the lasalocid biosynthetic pathway which involves characteristic aromatic ring formation and sequential cyclic ether formation. The finding of a thioesterase domain at the C-terminal of the seventh PKS indicates that intriguing oxidative cascade cyclization would occur after cleavage of the polyketide intermediate from PKS. Based on this observation, we have recently reported the enzymatic transformation of a bisepoxide intermediate to lasalocid with the recombinant epoxide hydrolase, Lsd19.

Analysis of specific mutants in the lasalocid gene cluster: evidence for enzymatic catalysis of a disfavoured polyether ring closure.

Lasalocid is a highly atypical polyether ionophoric antibiotic, firstly because it contains a type of aromatic ring normally associated with fungal polyketides, and secondly because the formation of its tetrahydropyran ring appears to contravene Baldwin's rules, which predict the kinetically preferred routes for cyclisation reactions in organic chemistry. The lasalocid biosynthetic gene cluster has been cloned from Streptomyces lasaliensis, and the las locus (73,533 bp) was found to contain seven modular polyketide synthase (PKS) genes, including all the activities necessary for the synthesis of the aromatic moiety. Specific deletion from the gene cluster of the flanking lasC gene, which is predicted to encode a flavin-linked epoxidase, abolished production both of lasalocid and of the minor cometabolite iso-lasalocid without leading to accumulation of an identifiable intermediate; this suggests that oxidative cyclisation to form the polyether rings takes place on the PKS before release of the full-length polyketide product. Meanwhile, a mutant in which the adjacent epoxide hydrolase lasB had been deleted produced iso-lasalocid only. Iso-lasalocid differs from lasalocid in the replacement of the tetrahydropyran ring by a tetrohydrofuran ring and represents the kinetically favoured product of cyclisation. The LasB epoxide hydrolase is therefore directly implicated in control of the stereochemical course of polyether ring formation during lasalocid biosynthesis.

Epoxide hydrolase Lsd19 for polyether formation in the biosynthesis of lasalocid A: direct experimental evidence on polyene-polyepoxide hypothesis in polyether biosynthesis.

Polyether metabolites are an important class of natural products. Although their biosynthesis, especially construction of polyether skeletons, attracted organic chemists for many years, no experimental data on the enzymatic polyether formation has been obtained. In this study, a putative epoxide hydrolase gene lsd19 found on the biosynthetic gene cluster of an ionophore polyether lasalocid was cloned and successfully overexpressed in Escherichia coli. Using the purified Lsd19, a proposed substrate, bisepoxyprelasalocid, and its synthesized analogue were successfully converted into lasalocid A and its derivative via a 6-endo-tet cyclization mode. On the other hand, treatment of the bisepoxide with trichloroacetic acid gave isolasalocid A via a 5-exo-tet cyclization mode. Therefore, the enzymatic conversion observed in this study unambiguously showed that the bisepoxyprelasalocid is an intermediate of the lasalocid biosynthesis and that Lsd19 catalyzes the sequential cyclic ether formations involving an energetically disfavored 6-endo-tet cyclization. This is the first example of the enzymatic epoxide-opening reactions leading to a polyether natural product.

Frequent loss and restoration of antibiotic production by Streptomyces lasaliensis.

Antibiotic nonproducing variants of Streptomyces lasaliensis NRRL 3382R, which makes the polyether antibiotic lasalocid A (Las) and the quinoxaline antibiotic echinomycin (Ech), arose at a frequency of 3-11% after treatment with three different mutagens or regeneration of protoplasts compared with a spontaneous frequency of less than 0.1%. Cosynthesis of lasalocid A was not observed upon testing a large number of Las- mutants in different pair-wise combinations, nor did these mutants accumulate probable intermediates of lasalocid A biosynthesis. These results suggest that loss of the las genes or their expression is induced at a high frequency by mutagenic treatments. In fusions of protoplasts of a strain with the las+ ech+ spo+ nic-1 rif-3 markers with strains bearing the Las- LasS Ech- Bld- (or spo+) str-1 markers, Las+ Ech+ Spo+ StrR progeny were produced at a 61-89% frequency compared with a 1-9% frequency of StrR antibiotic producing progeny with the nic-1 or rif-3 genotypes. The more frequent restoration of antibiotic production than prototrophy or rifampicin sensitivity indicates that these antibiotic characters did not behave as normal chromosomal markers. Therefore the genetic instability might be due to the involvement of a plasmid in antibiotic production. The apparent lack of infectious transfer of the Las+ character to Las- parents in conjugal matings between the few strains tested and no correlation between the presence of a large plasmid, pKSL, and lasalocid A production in several strains of S. lasaliensis do not favor the latter hypothesis, but they do not conclusively disprove it. Consequently, we suggest that a plasmid or another mobile genetic element is controlling antibiotic production in S. lasaliensis.

Biosynthesis of lasalocid A: biochemical mechanism for assembly of the carbon framework.

Labeling experiments on the biosynthesis of the polyether antibiotic lasalocid A (1) using carboxylic acid precursors bearing 13C, 2H, and 3H labels at various positions established the following: (1) 2H or 3H at C-2 of propionate or 2H at C-2 of butyrate was partially retained at C-12 and C-14 of 1, respectively. (2) 2H at C-2 of propionate or at C-2 and C-3 of succinate did not label C-10. These and earlier data [Hutchinson, C. R., Sherman, M. M., Vederas, J. C., & Nakashima, T. T. (1981) J. Am. Chem. Soc. 103, 5953; Hutchinson, C. R., Sherman, M. M., McInnes, A. G., Walter, J. A., & Vederas, J. C. (1981) J. Am. Chem. Soc. 103, 5956] are consistent with a hypothesis for the stereochemical control of lasalocid A biosynthesis, whose main tenets are that the configuration of C-12 and C-14 is determined by the stereoselectivity of the carbon chain forming condensation between acyl thio ester and 2-carboxyacyl thio ester intermediates and that the configuration of C-11 and C-15 results from the reduction of 2-keto thio ester intermediates with opposing stereospecificities.

Biosynthesis of lasalocid A. Biochemical alteration of polyether antibiotic production.

The effect of known precursors, their fluorinated analogs, and biochemical inhibitors on the production of the polyether antibiotic, lasalocid A (1), by resting cells of Streptomyces lasaliensis was determined to study the biochemistry and regulation of antibiotic biosynthesis in vivo.

Biosynthesis of lasalocid A. Metabolic interrelationships of carboxylic acid precursors and polyether antibiotics.

The metabolic interrelationships of isobutyrate, n-butyrate, and propionate in Streptomyces lasaliensis are established to show how these acids are used as precursors for the biosynthesis of the polyether antibiotic, lasalocid A.