Dissecting the enzymology of complex natural products: Main case study SgcA1, a thymidylyltransferase.

摘要

Some of the most powerful anti-tumor agents to date belong to the enediyne family. The most potent member of this family is C-1027, produced by Streptomyces globisporus. C-1027 consists of four building blocks, one of which is a functionalized sugar, 4-deoxy-4-(dimethylamino)-5,5,-dimethyl-D-ribopyranose. On the basis of sequence analysis it was predicted that the enzyme of interest, SgcA1, catalyzes the first step towards the synthesis of the functionalized sugar of C-1027. It activates glucose-alpha-1-phosphate by converting it to thymidine diphosphate-alpha-D-glucose, thus diverting the sugar precursor into secondary metabolite biosynthesis.;The aim of this work is to elucidate the mechanism of SgcA1 through synthetic, biochemical, and molecular biology methods. Characterization of SgcA1 with its natural substrates was carried out and kinetic parameters established. A rationally designed inhibitor was proposed and successfully synthesized. Its inhibition constant was calculated through a spectrophotometric assay. SgcA1 was screened for crystallization conditions and they have been identified.;Along with SgcA1, there have been attempts at elucidating the structure of several other proteins. These include SgcA6 also from the C-1027 biosynthetic pathway, LmnQ from the leinamycin biosynthetic pathway, NcsC3 and NcsC6 from the neocarzinostatin biosynthetic pathway.