L-Cysteine (Cys) is an essential building block for the biosynthesis of new proteins and serves as a precursor for several biologically important sulfur-containing molecules, such as coenzyme A, taurine, glutathione, and inorganic sulfate. However, organisms must tightly regulate the concentration of exogenous Cys, as elevated levels of this semi-essential amino acid can be extremely harmful. The non- heme iron enzyme cysteine dioxygenase (CDO) serves to maintain the proper Cys levels by catalyzing the oxidation of Cys to cysteine sulfinic acid. Malfunctioning of CDO and the consequent accumulation of Cys have been linked to several neurodegenerative diseases. The decarboxylation of Cys during coenzyme A synthesis generates cysteamine. The constitutive degradation of coenzyme A releases this cysteamine moiety, which can be converted to hypotaurine by the non-heme iron enzyme cysteamine (2-aminoethanethiol) dioxygenase (ADO). Hypotaurine is subsequently oxidized to taurine, an amino thiol acid that plays numerous important roles in mammalian tissues, including maintaining cardiac functions, protecting neural cells from excitotoxicity and ischemia, serving as a neurotransmitter, and stabilizing skeletal muscle membrane. Despite catalyzing the oxidation of two structurally similar thiol compounds, CDO and ADO show very inefficient cross-utilization of substrates. By studying CDO and ADO in parallel, we are presented with an opportunity to conclusively determine the substrate selectivity mechanism each enzyme employs, and thus how Cys and cysteamine levels may be independently regulated in vivo. The overall objective of the research outlined in this proposal is, therefore, to identify the roles of key amino acid residues with regards to substrate selectivity, positioning, and activation in the CDO and ADO catalytic mechanisms. With this objective in mind, we have devised the following Specific Aims: 1. Elucidate structure/function relationships in the catalytic mechanism of CDO. 2. Assess the effects of differences in key conserved amino acid residues between eukaryotic and prokaryotic CDOs on the nature of active site/substrate interactions. 3. Establish the order and modes by which the substrates cysteamine and O2 bind to the ADO active site and the mechanism of thiol oxidation. 4. Explore the geometric/electronic structures and reaction mechanisms of CDO and ADO mimics. To accomplish these aims, we will employ a combination of biochemical, spectroscopic, and computational tools for studying the resting states and substrate (analogue) adducts of the native enzymes, select variants, and small-molecule functional CDO and ADO mimics.
Cysteine dioxygenase and cysteamine dioxygenase, which are the only known mammalian thiol dioxygenases, play essential roles in the hypotaurine/taurine biosynthesis. The malfunctioning of these enzymes has been implicated in several neurodegenerative diseases (e.g., Parkinson?s, Alzheimer?s, and motor neuron diseases), as well as autoimmune disorders. The research outlined in this proposal will provide unprecedented insights into the structure/function relationships in these enzymes and allow us to conclusively determine the substrate selectivity mechanism that each employs, and thus how cysteine and cysteamine levels may be independently regulated in vivo.
|Fischer, Anne A; Stracey, Nuru; Lindeman, Sergey V et al. (2016) Synthesis, X-ray Structures, Electronic Properties, and O2/NO Reactivities of Thiol Dioxygenase Active-Site Models. Inorg Chem 55:11839-11853|