Sequence analysis of the biosynthetic gene cluster for the enediyne antitumor antibiotic C-1027 from Streptomyces globisporus has previously suggested that the sgcA1 gene encodes a alpha-d-glucopyranosyl-1-phosphate thymidylyltransferase (Glc-1-P-TT) catalyzing the first step in the biosynthesis of the 4-deoxy-4-(dimethylamino)-5,5-dimethyl-d-ribopyranose moiety by activating alpha-d-glucopyranosyl-1-phosphate (Glc-1-P) into deoxythymidine diphosphate-alpha-d-glucose (dTDP-Glc). Here we report the overexpression of sgcA1 in E. coli, purification of the overproduced SgcA1 to homogenetity, biochemical and kinetic characterization of the purified SgcA1 as a Glc-1-P-TT, and yield improvement for C-1027 production by overexpression of sgcA1 and its flanking gene in S. globisporus. These findings provide biochemical evidence supporting the genetics-based hypothesis for C-1027 biosynthesis, set the stage for further investigation of the deoxysugar biosynthetic pathway, and demonstrate the utility of sugar biosynthesis genes in natural product yield improvement via combinatorial biosynthesis methods. In contrast to the homotetrameric quaternary structure known for Glc-1-P-TT enzymes from primary metabolic pathways, Glc-1-P-TT enzymes such as SgcA1 from secondary metabolic pathways are monomeric in solution. Sequence differences between the two subclasses of Glc-1-P-TT enzymes were noted. The monomeric structural feature of the latter enzymes could be exploited in engineering Glc-1-P-TT enzymes with broad substrate specificity for structural diversity via the glycorandomization strategy.