Nodusmicin

A number of structurally diverse natural products harboring pyrrole moieties possess a wide range of biological activities. Studies on biosynthesis of pyrrole ring have shown that pyrrole moieties are derived from L-proline. Nargenicin A(1), a saturated alicyclic polyketide from Nocardia sp. CS682, is a pyrrole-2-carboxylate ester of nodusmicin. We cloned and identified a set of four genes from Nocardia sp. CS682 that show sequence similarity to the respective genes involved in the biosynthesis of the pyrrole moieties of pyoluteorin in Pseudomonas fluorescens, clorobiocin in Streptomyces roseochromogenes subsp. Oscitans, coumermycin A(1) in Streptomyces rishiriensis, one of the pyrrole rings of undecylprodigiosin in Streptomyces coelicolor, and leupyrrins in Sorangium cellulosum. These genes were designated as ngnN4, ngnN5, ngnN3, and ngnN2. In this study, we presented the evidences that the pyrrole moiety of nargenicin A(1) was also derived from L-proline by the coordinated action of three proteins, NgnN4 (proline adenyltransferase), NgnN5 (proline carrier protein), and NgnN3 (flavine-dependent acyl-coenzyme A dehydrogenases). Biosynthesis of pyrrole moiety in nargenicin A(1) is initiated by NgnN4 that catalyzes ATP-dependent activation of L-proline into L-prolyl-AMP, and the latter is transferred to NgnN5 to create prolyl-S-peptidyl carrier protein (PCP). Later, NgnN3 catalyzes the two-step oxidation of prolyl-S-PCP into pyrrole-2-carboxylate. Thus, this study presents another example of a pyrrole moiety biosynthetic pathway that uses a set of three genes to convert L-proline into pyrrole-2-carboxylic acid moiety.

Coloradocin was isolated from a fermentation broth by adsorption onto Amberlite XAD-2. The activity was eluted in MeOH and purified by gel filtration on Shephadex LH-20, followed by liquid-liquid chromatography on diol-bonded silica gel. The last two steps in the purification of this antibiotic included reverse-phase chromatography on C18-bonded silica gel and countercurrent chromatography on an Ito Coil Planet Centrifuge to give material of 90% purity. Analytically pure material was obtained by preparative HPLC. As a result of extensive homo and heteronuclear two-dimensional NMR studies, a structure was proposed for coloradocin. The structure consists of a decalin ring system fused to a 10-membered macrolactone which in turn is fused to a 14-membered macrolactone possessing an enol ether in conjugation with an unsaturated cyclic anhydride functionality. Coloradocin is related to a small class of antibiotics which include nodusmicin and nargenicin and was shown to be identical to luminamicin for which no structure has been reported.

The absolute stereostructure of luminamicin, an anaerobic antibiotic, has been determined by using conformational analysis via high-temperature molecular dynamics, NMR spectroscopy, and the modified Mosher method. It was found that luminamicin has the S, S, R, R, R, R, S, S, S, R, and S configurations at C2, C4, C7, C9, C10, C11, C12, C13, C16, C28, and C29, respectively. This configuration is the same as that found in nodusmicin, which has a chemical structure quite similar to luminamicin. The structure of luminamicin consists of three different rings, i.e., a decalin ring, a 10-membered macrolactone ring, and a 14-membered macrolactone ring. The resulting three-dimensional structure of luminamicin shows an interesting feature in that the maleic anhydride functionality in conjugation with the enol ether group of the 14-membered macrolactone is nearly perpendicular to the plane of the other two rings.