Various ATPases have been tested for their sensitivity to naturally occurring unusual macrolides and their chemically modified derivatives, which are structurally related to bafilomycin A1 (1), the first specific inhibitor of vacuolar ATPases. The structure-activity study showed that in general the concanamycins, 18-membered macrolides, are better and more specific inhibitors than the bafilomycins of this class of membrane-bound ATPases. The additional carbohydrate residue is not responsible for the improved activity. The importance of an intact hemiketal ring, which is part of an intramolecular hydrogen-bonding network, and the effects of the size of the macrolactone ring are discussed. The structurally related elaiophylin (13), a C2-symmetric macrodiolide antibiotic, proved to be inactive on vacuolar ATPases but still retained its inhibitory effect on P-type ATPases.

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Two genes (mtmD and mtmE) were cloned and sequenced from the mithramycin producer Streptomyces argillaceus. Comparison with proteins in databases and enzymatic assays after expression in Escherichia coli showed that they encode a glucose-1-phosphate:TTP thymidylyl transferase and a TDP-D-glucose 4,6-dehydratase, respectively. The mtmD gene was inactivated by gene replacement, generating a nonproducing mutant that accumulates a tetracyclic compound designated premithramycinone. The identification of premithramycinone reveals new aspects of the mithramycin biosynthetic pathway and suggests that at least some glycosylations occur before breakage of the fourth ring.Mithramycin (also designated aureolic acid, plicamycin, mithracin, LA7017, and A2371) is an antitumor drug synthesized by different actinomycete species and has clinical application in the treatment of several tumors (14). It also possesses antibiotic activity against gram-positive but not gram-negative bacteria. Structurally, it is an aromatic polyketide containing a three-ring chromophoric aglycon derived from the condensation of 10 acetates (11). Genes for a type II polyketide synthase were cloned and sequenced from a mithramycin producer, Streptomyces argillaceus; their involvement in mithramycin biosynthesis has been demonstrated by insertional inactivation (9), and evidence in favor of the biosynthesis of the aglycon through a single polyketide backbone instead of two has been provided elsewhere (3). The mithramycin aglycon (mithramycinone) is glycosylated at positions 6 and 2 with disaccharide and trisaccharide moieties, respectively. These sugars belong to the large family of the 6-deoxyhexoses (6DOHs), which are synthesized through biosynthetic pathways that have early biosynthetic steps in common (8, 12). In 6DOH biosynthesis, first glucose-1-phosphate is converted into TDP-D-glucose by the action of a glucose-1-phosphate:TTP thymidylyl transferase. Then, a TDP-D-glucose 4,6-dehydratase is responsible for the conversion of TDP-D-glucose into TDP-D-4-keto-6-deoxyglucose. Further action of a 3,5-epimerase can direct TDP-D-4-keto-6-deoxyglucose into the L series of 6DOHs; in contrast, if there is no participation of an epimerase, the final sugars will be D-6DOHs. Further modifications in both L-and D-6DOHs via isomerization, methylation, reduction, dehydration, etc., can produce a great variety of 6DOHs, which are part of the structures of different actinomycete metabolites (12, 18).Here we report the cloning, sequencing, and heterologous expression of two genes involved in the biosynthesis of the sugar moieties of the antitumor drug mithramycin. These genes (mtmD and mtmE) encode a glucose-1-phosphate:TTP thymidylyl transferase and a TDP-D-glucose 4,6-dehydratase, respectively. We also report the construction of a non-mithra-FIG. 1. Schematic representation of the region sequenced from cosAR7 and the different deduced ORFs and its location with respect to the mithramycin polyketide synthase (PKS). The bar indicates the region showing homology to t...

Bioassay-guided fractionation of an extract of Holarrhena floribunda stem, has led to the isolation of the new trichothecenes, 8-dihydrotrichothecinol A (1), loukacinol A (2), and loukacinol B (3), and the known compounds, trichothecolone (4), trichothecin (5), trichothecinol A (6), rosenonolactone (7), 6beta-hydroxyrosenonolactone (8), and rosololactone (9). The structures were determined by spectral and chemical methods, and absolute configurations were established by a modified Horeau's method using HPLC. Compounds 1 and 6 exhibited significant cytotoxicity against several human tumor cell lines, whereas compound 8 showed moderate and weak antileishmanial activity toward extracellular and intracellular Leishmania donovani, respectively.