Mechanistic Investigation of Isonitrile Formation Catalyzed by the Nonheme Iron/α-KG-Dependent Decarboxylase (ScoE)

2020-02-20T20:12:36Z (GMT) by Hong Li Yongjun Liu
Recent structural and biochemical evidence showed that ScoE from Streptomyces coeruleorubidus is a nonheme iron/α-KG-dependent decarboxylase, which catalyzes the formation of isonitrile group by desaturation and decarboxylation. This discovery offers an alternative mechanism for isonitrile formation. The other isonitrile synthases, such as IsnA, XnPvcA, or AmbI1/AmbI2, convert R–CH­(−NH2)–CO2 to R–CH­(−NC)–CO2 by introducing an additional carbon unit; however, ScoE catalyzes the conversion of R–NH–CH2–CO2 to R–NC through oxidative decarboxylation. To explore the catalytic mechanism of ScoE, on the basis of the high-resolution crystal structure, the enzyme–substrate complex models were constructed and a series of combined QM/MM calculations were performed. Our results reveal that the ScoE-catalyzed reaction contains two decoupled parts, desaturation and decarboxylation. The FeIV-oxo-triggered desaturation includes two consecutive H-abstractions, which are similar to the C–C single bond desaturation catalyzed by other nonheme iron/α-KG-dependent desaturases. In the second stage reaction, the decarboxylation of the substrate radical generated by H-abstraction was calculated to be quite easy, whereas the previously proposed decarboxylation that involves the hydroxylated intermediate was calculated to be difficult. Importantly, the electron transfer from the substrate to the iron center is the key factor for lowering the barrier of decarboxylation. Thus, the central iron ion is not only responsible for H-abstraction but also acts as an electron relay station for decarboxylation. In addition, this electron transfer was found to be coupled with a proton transfer, in which R310 and the associated H-bonding network play a critical role. In general, the first C–N desaturation is the rate-limiting step of the whole catalysis with an overall energy barrier of 17.6 or 16.9 kcal/mol in two competitive pathways, qualitatively agreeing with the estimated free energy (17.9–18.1 kcal/mol) from experiments. These results may provide useful information for understanding the biosynthesis of isonitrile and the oxidative decarboxylation catalyzed by nonheme iron/α-KG-dependent enzymes.