Steady-state substrate specificity and O2-coupling efficiency of mouse cysteine dioxygenase

• Cysteine dioxygenase (CDO) catalyzes formation of several sulfinic acid products.
• Substrate-specificity (V/K) for CDO can be measured over 4-orders of magnitude.
• The minimal substrate for CDO must contain both amine and thiol functional groups.
• Substrate perturbations significantly impact oxidative coupling efficiency.

Cysteine dioxygenase (CDO) is a non-heme mononuclear iron enzyme that catalyzes the oxygen-dependent oxidation of l-cysteine (Cys) to produce l-cysteine sulfinic acid (CSA). Sequence alignment of mammalian CDO with recently discovered thiol dioxygenase enzymes suggests that the mononuclear iron site within all enzymes in this class share a common 3-His first coordination sphere. This implies a similar mechanistic paradigm among thiol dioxygenase enzymes. Although steady-state studies were first reported for mammalian CDO over 45 years ago, detailed analysis of the specificity for alternative thiol-bearing substrates and their oxidative coupling efficiencies have not been reported for this enzyme. Assuming a similar mechanistic theme among this class of enzymes, characterization of the CDO substrate specificity may provide valuable insight into substrate-active site intermolecular during thiol oxidation. In this work, the substrate-specificity for wild-type Mus musculus CDO was investigated using NMR spectroscopy and LC–MS for a variety of thiol-bearing substrates. Tandem mass spectrometry was used to confirm dioxygenase activity for each non-native substrate investigated. Steady-state Michaelis–Menten parameters for sulfinic acid product formation and O2-consumption were compared to establish the coupling efficiency for each reaction. In light of these results, the minimal substrate requirements for CDO catalysis and O2-activation are discussed.

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