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

The isonitrile moiety is an electron-rich function-ality that decorates various bioactive natural products isolated fromdiverse kingdoms of life. Isonitrile biosynthesis was restricted forover a decade to isonitrile synthases, a family of enzymes catalyzinga condensation reaction betweenL-Trp/L-Tyr and ribulose-5-phosphate. The discovery of ScoE, a non-heme iron(II) and alpha-ketoglutarate-dependent dioxygenase, demonstrated an alternativepathway employed by nature for isonitrile installation. Biochem-ical, crystallographic, and computational investigations of ScoEhave previously been reported, yet the isonitrile formationmechanism remains obscure. In the present work, we employedin vitro biochemistry, chemical synthesis, spectroscopy techniques,and computational simulations that enabled us to propose a plausible molecular mechanism for isonitrile formation. Ourfindingsdemonstrate that the ScoE reaction initiates with C5 hydroxylation of (R)-3-((carboxymethyl)amino)butanoic acid to generate1,which undergoes dehydration, presumably mediated by Tyr96 to synthesize2in a trans configuration. (R)-3-isocyanobutanoic acidisfinally generated through radical-based decarboxylation of2, instead of the common hydroxylation pathway employed by thisenzyme superfamily