Probing the Mechanism of Isonitrile Formation by a Non-Heme Iron(II)-Dependent Oxidase/Decarboxylase

The
isonitrile moiety is an electron-rich functionality that decorates
various bioactive natural products isolated from diverse kingdoms
of life. Isonitrile biosynthesis was restricted for over a decade
to isonitrile synthases, a family of enzymes catalyzing a condensation
reaction between l-Trp/l-Tyr and ribulose-5-phosphate.
The discovery of ScoE, a non-heme iron­(II) and α-ketoglutarate-dependent
dioxygenase, demonstrated an alternative pathway employed by nature
for isonitrile installation. Biochemical, crystallographic, and computational
investigations of ScoE have previously been reported, yet the isonitrile
formation mechanism remains obscure. In the present work, we employed
in vitro biochemistry, chemical synthesis, spectroscopy techniques,
and computational simulations that enabled us to propose a plausible
molecular mechanism for isonitrile formation. Our findings demonstrate
that the ScoE reaction initiates with C5 hydroxylation of (R)-3-((carboxymethyl)­amino)­butanoic acid to generate 1, which undergoes dehydration, presumably mediated by Tyr96
to synthesize 2 in a trans configuration. (R)-3-isocyanobutanoic acid is finally generated through radical-based
decarboxylation of 2, instead of the common hydroxylation
pathway employed by this enzyme superfamily.