Abstract
Cysteine dioxygenase (CDO) is a non-heme mono-iron enzyme, which catalyses the first step of cysteine metabolism in various species. It is known that malfunction of this enzyme has implications for health in mammals but despite extensive research over the past four decades, no consensus with respect to the kinetics and mechanism of CDO has been reached. An important factor is the presence of a naturally occurring post-translational modification between Cys93 and Tyr157 in the mammalian form of the enzyme, which enhances activity and requires the presence of cysteine and oxygen for its formation. Another factor is the reactivity of the substrate cysteine, which can be oxidised to various products in the absence of CDO and form disulfide adducts with free cysteine residues within the enzyme. These adducts are thought to have detrimental effects on CDO activity, and this is of particular importance when assessing CDO activity in vitro, where supraphysiological levels of cysteine substrate are present.
The aim of this work was to develop a method for CDO activity assays, which provided both analytical and structural data for the subsequent assessment of CDO kinetics and establishment of a kinetic profile. Analytical data had to encompass information about both enzymatic and non-enzymatic oxidation of cysteine. The methods used to obtain this information were high performance liquid chromatography in combination with evaporative light scattering detection. Structural information assessing the presence of the activity-enhancing crosslink was obtained using denaturing gel electrophoresis. The great advantage of the method developed here was that analytical and structural information was obtained from the same sample and thus allowed direct correlation between these aspects. The method was used to assess activity of recombinant CDO from rat and a bacterium. Rat CDO was expressed as wild-type and a mutant (C164S), which lacked a cysteine residue at the active site entrance and could therefore not form a disulfide at this position with exogenous cysteine.
Both types of rat CDO possessed the crosslink and were approximately 15-fold more active than bacterial CDO, which lacked this post-translational modification. Dependent on pH and the fraction of crosslinked enzyme present in an assay, rat CDO also had the ability to further increase activity through crosslink formation. This resulted in sigmoidal product formation curves during crosslink formation and indicated that crosslink formation possibly proceeded via an intermediate, which further increased enzyme activity.