The mtmVUC genes of the mithramycin gene cluster in Streptomycesargillaceus are involved in the biosynthesis of the sugar moieties

Abstract

Mithramycin is a glycosylated aromatic polyketide produced by Streptomyces argillaceus, and is used as an antitumor drug. Three genes (mtmV, mtmU and mtmC) from the mithramycin gene cluster have been cloned, and characterized by DNA sequencing and by analysis of the products that accumulate in nonproducing mutants, which were generated by insertional inactivation of these genes. The mtm V gene codes for a 2,3-dehydratase that catalyzes early and common steps in the biosynthesis of the three sugars found in mithramycin (D-olivose, D-oliose and D-mycarose); its inactivation caused the accumulation of the nonglycosylated intermediate premithramycinone. The mtmU gene codes for a 4-ketoreductase involved in D-oliose biosynthesis, and its inactivation resulted in the accumulation of premithramycinone and premithramycin A , the first glycosylated intermediate which contains a D-olivose unit. The third gene, mtmC, is involved in D-mycarose biosynthesis and codes for a C-methyltransferase. Two mutants with lesions in the mtmC gene accumulated mithramycin intermediates lacking the D-mycarose moiety but containing D-olivose units attached to C-12a in which the 4-keto group is unreduced. This suggests that mtmC could code for a second enzyme activity, probably a D-olivose 4-ketoreductase, and that the glycosyltransferase responsible for the incorporation of D-olivose (MtmGIV) shows some degree of flexibility with respect to its sugar co-substrate, since the 4-ketoanalog is also transferred. A pathway is proposed for the biosynthesis of the three sugar moieties in mithramycin.

Combinatorial Biosynthesis of Antitumor Deoxysugar Pathways in Streptomyces griseus : Reconstitution of “Unnatural Natural Gene Clusters” for the Biosynthesis of Four 2,6- d -Dideoxyhexoses

“…Therefore, the structure of 8-demethyl-8-␤-Dboivinosyl-tetracenomycin C could be deduced for DBOV-TCMC. This was fully confirmed by the 13 C NMR data (Table 3).…”

“…The observed stereochemistry at the 3Ј position (a multiple resulting from three small 3 J H-H couplings with neighboring protons reveals an axially attached OH group) and in the 4Ј position (dd, J ϭ 9, 3 Hz, revealing an equatorially attached OH group) is consistent with digitoxose stereochemistry. This suggests the structure of 8-demethyl-8-␤-D-digitoxosyltetracenomycin C for DDIG-TCMC, which was fully confirmed by the 13 C NMR data (Table 2).…”

“…However, when pMP1*UII was introduced into S. lividans 16F4, no oliosyl-tetracenomycin or glycosylated tetracenomycin was observed (data not shown). In order to prove the functionality of pMP1*UII, i.e., its capability to direct the biosynthesis of Doliose, we expressed the plasmid in Streptomyces argillaceus M7U1, a mithramycin nonproducer mutant in which D-oliose biosynthesis is affected (13). Complementation of this mutant was achieved, indicating that D-oliose biosynthesis was recovered and therefore that genes in pMP1*UII were functional (data not shown).…”

“…However, D-oliose, and especially D-digitoxose and D-boivinose, are quite unusual among bioactive compounds produced by microorganisms. Genes involved in the biosynthesis of D-olivose and D-oliose have been identified from different antibiotic producer microorganisms (9,13,15,16,20,24,40,42,43). Very recently, a gene cluster for the biosynthesis of D-digitoxose was also identified (5).…”

Chemical Diversity of Polyene Macrolides Produced by Streptomyces noursei ATCC 11455 and Recombinant Strain ERD44 with Genetically Altered Polyketide Synthase NysC

“…However, L-or D-mycarose has been found in other polyketide antibiotic molecules with antibacterial activities, such as mithramycin A (25,48), tylosin (4), and platenolide glycosides isolated from S. hygroscopicus (26). Studies of erythromycin A biosynthesis also showed the presence of L-mycarose in some intermediates (erythromycins C and D), which is later converted to L-cladinose (44).…”