Key result
In vitro reconstitution of the dTDP-l-daunosamine biosynthetic pathway identified a major bottleneck and demonstrated the potential for enzymatic synthesis of tailored nucleotide-activated sugars.
Why the study?
Sugar moieties like L-daunosamine are key to anthracycline bioactivity and toxicity, but the enzymatic assembly of the precursor dTDP-L-daunosamine needed further characterization.
The in vitro reconstitution of the dTDP-l-daunosamine pathway provides a platform for understanding anthracycline glycosylation and generating novel glycosylated derivatives with potentially lower cardiotoxicity.
May support engineering of anthracycline analogs; leaves open scalability and clinical translation.
High Resolution Image Download MS PowerPoint Slide Many small molecule natural products are decorated with sugar moieties that are essential for their biological activity. A considerable number of natural product glycosides and their derivatives are clinically important therapeutics. Anthracyclines like daunorubicin and doxorubicin are examples of valuable glycosylated natural products used in medicine as potent anticancer agents. The sugar moiety, l -daunosamine (a highly modified deoxyhexose), plays a key role in the bioactivity of these molecules as evidenced by semisynthetic anthracycline derivatives such as epirubicin, wherein alteration in the configuration of a single stereocenter of the sugar unit generates a chemotherapeutic drug with lower cardiotoxicity. The nucleotide activated sugar donor that provides the l -daunosamine group for attachment to the natural product scaffold in the biosynthesis of these anthracyclines is dTDP- l -daunosamine. In an in vitro system, we have reconstituted the enzymes in the daunorubicin/doxorubicin pathway involved in the biosynthesis of dTDP- l -daunosamine. Through the study of the enzymatic steps in this reconstituted pathway, we have gained several insights into the assembly of this precursor including the identification of a major bottleneck and competing reactions. We carried out kinetic analysis of the aminotransferase that catalyzes a limiting step of the pathway. Our in vitro reconstituted pathway also provided a platform to test the combinatorial enzymatic synthesis of other dTDP-activated deoxyhexoses as potential tools for “glycodiversification” of natural products. To this end, we replaced the stereospecific ketoreductase that acts in the last step of dTDP- l -daunosamine biosynthesis with an enzyme from a heterologous pathway with opposite stereospecificity and found that it is active in the in vitro pathway, demonstrating the potential for the enzymatic synthesis of nucleotide-activated sugars with regio- and stereospecific tailoring.
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Mohideen et al. (2021) studied this question. In vitro reconstitution of the dTDP-l-daunosamine biosynthetic pathway was evaluated on Enzymatic steps and kinetic analysis of the aminotransferase in the dTDP-l-daunosamine pathway. In vitro reconstitution of the dTDP-l-daunosamine biosynthetic pathway identified a major bottleneck and demonstrated the potential for enzymatic synthesis of tailored nucleotide-activated sugars.
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