Sulfur is an essential element found ubiquitously in living systems. However, there exist only a few sulfur-containing sugars in nature and their biosyntheses have not been studied. BE-7585A produced by Amycolatopsis orientalis subsp. vinearia BA-07585 has a 2-thiosugar and is a member of the angucycline class of compounds. We report herein the results of our initial efforts to study the biosynthesis of BE-7585A. Spectroscopic analyses verified the structure of BE-7585A, which is closely related to rhodonocardin A. Feeding experiments using 13 C-labeled acetate were carried out to confirm that the angucycline core is indeed polyketide-derived. The results indicated an unusual manner of angular tetracyclic ring construction, perhaps via a Baeyer-Villiger type rearrangement. Subsequent cloning and sequencing led to the identification of the bex gene cluster spanning ~30 kbp. A total of 28 open reading frames, which are likely involved in BE-7585A formation, were identified in the cluster. In view of the presence of a homologue of a thiazole synthase gene (thiG), bexX, in the bex cluster, the mechanism of sulfur incorporation into the 2-thiosugar moiety could resemble that found in thiamin biosynthesis. A glycosyltransferase homologue, BexG2, was heterologously expressed in E. coli. The purified enzyme successfully catalyzed the coupling of 2-thioglucose 6-phoshate and UDP-glucose to produce 2-thiotrehalose 6-phosphate, which is the precursor of the disaccharide unit in BE-7585A. On the basis of these genetic and biochemical experiments, a biosynthetic pathway for BE-7585A can now be proposed. The combined results set the stage for future biochemical studies of 2-thiosugar biosynthesis and BE-7585A assembly.The angucycline class of natural products, which have a characteristic fused four-ring frame assembled in an angular manner, are rich in antibacterial or anticancer activities. 1 BE-7585A (1), produced by Amycolatopsis orientalis subsp. vinearia BA-07585, is an angucycline-type natural product that inhibits thymidylate synthase. 2 In addition to the benz [a]anthraquinone core, BE-7585A also contains the deoxysugar, rhodinose (2), and a disaccharide appendage (4) containing a highly unusual 2-thioglucose (3). While sulfur is an essential element found ubiquitously in living systems, including amino acids, nucleic acids, metal clusters, enzyme cofactors, and many secondary metabolites, it is seldom found in carbohydrates. The rareness of the thiosugar entity prompted us to explore the biosynthesis of BE-7585A, especially the mode of sulfur incorporation into the 2-thiosugar moiety (3). Mechanistic details of several biological sulfur insertion reactions have recently been unraveled. 3 Highly regulated reactive intermediates such as protein persulfide, protein thiocarboxylate or S-adenosylmethioninedependent radical are often generated in the enzymes carrying out such reactions. Whether the same or different mechanisms are used to form the C-S bond in 2-thiosugar biosynthesis is the focus of this study.* T...