Leimkuhler Catherine, Fridman Micha, Lupoli Tania, Walker Suzanne, Walsh Christopher T, Kahne Daniel
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
J Am Chem Soc. 2007 Aug 29;129(34):10546-50. doi: 10.1021/ja072909o. Epub 2007 Aug 8.
The tetracyclic core of anthracycline natural products with antitumor activity such as aclacinomycin A are tailored during biosynthesis by regioselective glycosylation. We report the first synthesis of TDP-L-rhodosamine and demonstrate that the glycosyltransferase AknS transfers L-rhodosamine to the aglycone to initiate construction of the side-chain trisaccharide. The partner protein AknT accelerates AknS turnover rate for L-rhodosamine transfer by 200-fold. AknT does not affect the Km but rather affects the kcat. Using these data, we propose that AknT causes a conformational change in AknS that stabilizes the transition state and ultimately enhances transfer. When the subsequent glycosyltransferase AknK and its substrate TDP-L-fucose are also added to the aglycone, the disaccharide and low levels of a fully reconstituted trisaccharide form of aclacinomycin are observed.
具有抗肿瘤活性的蒽环类天然产物(如阿克拉霉素A)的四环核心在生物合成过程中通过区域选择性糖基化进行修饰。我们报道了TDP-L-罗糖胺的首次合成,并证明糖基转移酶AknS将L-罗糖胺转移至苷元上,从而启动侧链三糖的构建。伴侣蛋白AknT可使AknS将L-罗糖胺转移的周转速率提高200倍。AknT不影响米氏常数(Km),而是影响催化常数(kcat)。利用这些数据,我们提出AknT会使AknS发生构象变化,从而稳定过渡态并最终增强转移作用。当随后的糖基转移酶AknK及其底物TDP-L-岩藻糖也添加到苷元上时,可观察到二糖以及低水平的完全重构的阿克拉霉素三糖形式。