The effect of ten naturally occurring and two synthetic inhibitors of NADH :ubiquinone oxidoreductase (complex I) of bovine heart, Neurosporu crassa and Escherichia coli and g1ucose:ubiquinone oxidoreductase (glucose dehydrogenase) of Gluconobacter oxidans was investigated. These inhibitors could be divided into two classes with regard to their specifity and mode of action. Class I inhibitors, including the naturally occuring piericidin A, annonin VI, phenalamid A2, aurachins A and B, thiangazole and the synthetic fenpyroximate, inhibit complex I from all three species in a partially competitive manner and glucose dehydrogenase in a competitive manner, both with regard to ubiquinone. Class I1 inhibitors including the naturally occuring rotenone, phenoxan, aureothin and the synthetic benzimidazole inhibit complex I from all species in an non-competitive manner, but have no effect on the glucose dehydrogenase. Myxalamid PI could not be classified as above because it inhibits only the mitochondrial complex I and in a competitive manner. All inhibitors affect the electron-transfer step from the high-potential iron-sulphur cluster to ubiquinone. Class I inhibitors appear to act directly at the ubiquinone-catalytic site which is related in complex I and glucose dehydrogenase.NADH : ubiquinone oxidoreductase, also known as respiratory complex I of mitochondria, transfers electrons from NADH to ubiquinone and links this process with translocation of protons across the inner membrane.

Deductions from the molecular analysis of the 65,000-bp stigmatellin biosynthetic gene cluster are reported. The biosynthetic genes (stiA-J) encode an unusual bacterial modular type I polyketide synthase (PKS) responsible for the formation of this aromatic electron transport inhibitor produced by the myxobacterium Stigmatella aurantiaca. Involvement of the PKS gene cluster in stigmatellin biosynthesis is shown using site-directed mutagenesis. One module of the PKS is assumed to be used iteratively during the biosynthetic process, which seems to involve an unusual transacylation of the biosynthetic intermediate from an acyl carrier protein domain back to the preceding ketosynthase domain. Finally, the polyketide chain which is presumably catalyzed by a novel C-terminal domain in StiJ that does not resemble thioesterases, is cyclized and aromatized. The presented results of feeding experiments are in good agreement with the proposed biosynthetic scheme. In contrast to all other PKS type I systems reported to date, each module of StiA-J is encoded on a separate gene. The gene cluster contains a "stand alone" O-methyltransferase and two unusual O-methyltransferase domains embedded in the PKS. In addition, inactivation of a cytochrome P450 monooxygenase-encoding gene involved in post-PKS hydroxylation of the aromatic ring leads to the formation of two novel stigmatellin derivatives.

The biosynthetic mta gene cluster responsible for myxothiazol formation from the fruiting body forming myxobacterium Stigmatella aurantiaca DW4/3-1 was sequenced and analyzed. Myxothiazol, an inhibitor of the electron transport via the bc 1 -complex of the respiratory chain, is biosynthesized by a unique combination of several polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS), which are activated by the 4-phosphopantetheinyl transferase MtaA. Genomic replacement of a fragment of mtaB and insertion of a kanamycin resistance gene into mtaA both impaired myxothiazol synthesis. Genes mtaC and mtaD encode the enzymes for bis-thiazol(ine) formation and chain extension on one pure NRPS (MtaC) and on a unique combination of PKS and NRPS (MtaD). The genes mtaE and mtaF encode PKSs including peptide fragments with homology to methyltransferases. These methyltransferase modules are assumed to be necessary for the formation of the proposed methoxy-and ␤-methoxy-acrylate intermediates of myxothiazol biosynthesis. The last gene of the cluster, mtaG, again resembles a NRPS and provides insight into the mechanism of the formation of the terminal amide of myxothiazol. The carbon backbone of an amino acid added to the myxothiazolacid is assumed to be removed via an unprecedented module with homology to monooxygenases within MtaG.Myxobacteria are Gram-negative soil bacteria that are assigned to the two suborders Cystobacterineae and Sorangineae. Both belong to the ␦-group of the Proteobacteria (1). They are distinguished from most other bacteria by their ability to glide in swarms, to feed cooperatively, and to form fruiting bodies upon starvation (2, 3). In addition, they have been shown to produce a wide variety of secondary metabolites with unique structures and biological activities (for reviews, see Refs. 4 and 5). These include the electron transport inhibitors myxothiazol (6), stigmatellin (7), and myxalamids (5, 8) produced by different strains of Stigmatella aurantiaca (Cystobacterineae) and the epothilones produced by Sorangium cellulosum (Sorangineae) (9) (structures are given in Fig. 1). Due to their antitumor activity, epothilones have attracted great attention (10 -12). Myxothiazol as well as epothilones contain a thiazole ring that is formed by the incorporation of cysteine into the polyketide backbone (13). Thiazoline and thiazolidine structures of bacitracin in Bacillus licheniformis (14) and the bacterial siderophores yersiniabactin and mycobactin have recently been shown to be biosynthesized by a NRPS 1 or a combined PKS/NRPS in Yersinia pestis and Mycobacterium tuberculosis (15)(16)(17) . 22)). No such combinations have been published so far for the formation of a thiazole coupled to a polyketide structure. In addition to the bis-thiazole moiety, myxothiazol has some unique features: the unusual leucine derived starter unit 3-methyl-butyryl-CoA (13) and the linear polyketide backbone, which includes a ␤-methoxy-acrylate and a terminal amide structure.Little is known about the biochemistry o...

The aurachins, new quinoline alkaloids, were extracted with acetone from the biomass of the myxobacterium, Stigmatella aurantiaca strain Sg al5 and purified by columnchromatography. The four described aurachins A, B, C and D, were inhibitory for Gram-positive bacteria and a few yeasts and molds. They blocked NADH oxidation in beef heart submitochondrial particles.

The biosynthetic gene cluster of the myxochelin-type iron chelator was cloned from Stigmatella aurantiaca Sg a15 and characterized. This catecholate siderophore was only known from two other myxobacteria. The biosynthetic genes of 2,3-dihydroxybenzoic acid are located in the cluster (mxcC±mxcF ). Two molecules of 2,3-dihydroxybenzoic acid are activated and condensed with lysine in a unique way by a protein homologous to nonribosomal peptide synthetases (MxcG). Inactivation of mxcG, which encodes an adenylation domain for lysine, results in a myxochelin negative mutant unable to grow under iron-limiting conditions. Growth could be restored by adding Fe 31 , myxochelin A or B to the medium. Inactivation of mxcD leads to the same phenotype. A new type of reductive release from nonribosomal peptide synthetases of the 2,3-dihydroxybenzoic acid bis-amide of lysine from MxcG, catalyzed by a protein domain with homology to NAD(P) binding sites, is discussed. The product of a gene, encoding a protein similar to glutamate-1-semialdehyde 2,1-aminomutases (mxcL), is assumed to transaminate the aldehyde that is proposed as an intermediate. Further genes encoding proteins homologous to typical iron utilization and iron uptake polypeptides are reported.

The myxobacterium Stigmatella aurantiaca DW4/3-1 harbours an astonishing variety of secondary metabolic gene clusters, at least two of which were found by gene inactivation experiments to be connected to the biosynthesis of previously unknown metabolites. In this study, we elucidate the structures of myxochromides S1-3, novel cyclic pentapeptide natural products possessing unsaturated polyketide side chains, and identify the corresponding biosynthetic gene locus, made up of six nonribosomal peptide synthetase modules. By analyzing the deduced substrate specificities of the adenylation domains, it is shown that module 4 is most probably skipped during the biosynthetic process. The polyketide synthase MchA harbours only one module and is presumably responsible for the formation of the variable complete polyketide side chains. These data indicate that MchA is responsible for an unusual iterative polyketide chain assembly.

Microorganisms produce iron-chelating compounds to sequester the iron essential for growth from the environment. Many of these compounds are biosynthesized by nonribosomal peptide synthetases, some in cooperation with polyketide synthases. Myxochelins are produced by the myxobacterium Stigmatella aurantiaca Sg a15, and the corresponding gene cluster was cloned recently. We have undertaken to express heterologously the myxochelin biosynthetic machinery in Escherichia coli. To activate the involved proteins posttranslationally, they were coexpressed with the phosphopantetheinyltransferase MtaA from the myxothiazol biosynthetic gene cluster. Phosphopantetheinylation of the carrier proteins could be verified by protein mass analysis. Six active domains in proteins MxcE, MxcF, and MxcG are capable of assembling myxochelin from ATP, NAD(P)H, lysine, and 2,3-dihydroxybenzoic acid in vitro. This fact demonstrates that the condensation domain of MxcG performs two condensation reactions, creating the arylcapped ␣-amide and the aryl-capped ␥-amide of the molecule. A previously unknown type of reductive release is performed by the reduction domain of MxcG, which alternatively uses NADPH and NADH to set free the peptidyl-carrier protein-bound thioester as an aldehyde and further reduces it to the alcohol structure that can be found in myxochelin A. This type of reductive release seems to be a general mechanism in polyketide and nonribosomal peptide biosynthesis, because several systems with C-terminal similarity to the reductase domain of MxcG can be found in the databases. Alternatively, the aldehyde can be transaminated, giving rise to a terminal amine.

A bouquet of bacteria: Methylisoborneol (1) is a widely occurring volatile from bacteria and an undesirable flavor (off‐flavor) in the food industry. The analysis of isotopomers obtained by feeding isotopically labeled precursors to myxobacteria revealed the biosynthetic pathway to 1. Geranylpyrophosphate (GPP) is alkylated by S‐adenosylmethionine (SAM) and the product is cyclized to 1. The methylation of GPP is unprecedented in nature.