Elucidation of divergent desaturation pathways in the formation of vinyl isonitrile and isocyanoacrylate

We studied two bacterial pathways that synthesize vinyl isonitrile compounds and vinyl isocyanoacrylate compounds. Our crystal structure of enzyme explains the substrate scope of enzymes involved in these pathways.
Elucidation of divergent desaturation pathways in the formation of vinyl isonitrile and isocyanoacrylate
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Introducing a reactive chemical functional group to chemical synthon can greatly expand compound libraries in organic synthesis. In biological systems, vinyl isonitrile and isocyanoacrylate groups in natural products can be warheads to drive bioactivity, often used as antibacterial or antifungal reagents. Incorporating these groups into non-canonical acid derivatives can be used for imaging or target identification in cells.

However, chemical preparation of vinyl isonitrile and isocyanoacrylate-containing compounds usually involves sophisticated synthetic strategy. Fundamental understanding of how natural pathways produce these compounds provide insight to engineer and utilize them as powerful tools for producing valuable compounds in an efficient and sustainable manner.

In our recently published work in Nature Communications, we studied two bacterial pathways synthesizing vinyl isonitrile or isocyanoacrylate from tyrosine. Specifically, our work focused on PIsnB and PvcB, two iron- and 2-oxoglutarate-dependent (Fe/2OG) enzymes involved in two different types of desaturations to produce building blocks in many isonitrile-containing natural products. Understanding this step reveals how enzyme potentially control reaction selectivity and substrate promiscuity, and further give us great opportunities to expand the natural product collections.

To understand how PIsnB and PvcB capture the substrate and carry out the desaturation reaction, we crystallized one of the enzymes, PIsnB, and obtained the enzyme structure complexed with its substrate. Our enzyme structure explains how both PIsnB and PvcB precisely bring targeted hydrogen atom of the substrate near the metal center for C-H activation while preventing isonitrile chelation to the metal ion center. Moreover, we demonstrated that the hydroxylated compound, a canonical reactivity for Fe/2OG enzymes, is not an intermediate for desaturation. Instead, following hydrogen atom abstraction (HAT), the formation of a benzylic carbocation is more likely deployed to construct vinyl isonitrile and isocyanoacrylate moieties. This observation is further supported by the in vitro results using fluorinated-probe. 

Figure 1. Substrate recognition mode of PIsnB is revealed by X-ray crystallography. Two homologs of Fe/2OG dependent enzymes (PvcB & PIsnB) differ in their product profile. Probable mechanisms are proposed based on X-ray crystallography and in vitro enzyme assays using mechanistic probes.

These discoveries provide foundations for engineering. In vitro enzyme assays show that both PIsnB and PvcB have flexible substrate scope whereas they can accommodate modified substrates. More interestingly, the reactivity of the enzyme can be tuned. The desaturation reroutes to the hydroxylation when the potential carbocation is destabilized, such as substituting the hydroxyl group of tyrosine with an electron-withdrawing group. Moreover, bioinformatics studies reveal that these enzymes contain divergent residues at conserved positions. Although further studies are still ongoing to switch their reactivity to the other, our sequence comparison provide a general guide to forecast the reaction selectivity between the formation of vinyl isonitrile or isocyanoacrylate moiety. Taken together, our findings shed light on the reaction mechanism of Fe/2OG enzyme catalyzed desaturation and further highlights the chemical and engineering potential for Fe/2OG-dependent enzymes.

For the full content of the research, please check the article published on Nature Communications (Link).

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Chemistry
Physical Sciences > Chemistry

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