Associate Professor, Department of Pharmacology and Toxicology
University of Kansas
Effect of Methionine Substitution and Oxidation on the Structure-Function of COMT (2015-2016)
Oxidation of methionine residues of the enzyme catechol-O-methyltransferase (COMT) enzyme causes reduction in its ability to degrade dopamine. Determining the effect of methionine oxidation on COMT’s structure may lead to novel therapeutic treatments aiming at protecting from this modification that may be more prevalent in neurological disorders.
The objective of this project is to understand the effect of methionine residues and their oxidation on the structure and function of the dopamine (DA)-metabolic enzyme catechol-O-methyltransferase (COMT). The human COMT gene features a single-nucleotide polymorphism at amino acid 158 of the membrane bound COMT and at amino acid 108 of the soluble COMT, consisting in the substitution of a valine (Val) residue with methionine (Met). This substitution leads to a marked reduction of COMT’s enzymatic activity and higher cortical DA levels. Studies have shown high schizophrenia vulnerability in homozygous carriers for either Val or Met variants who abused cannabis in adolescence. In recent studies, we showed that the reduction in COMT activity in the Met variant is dependent on the oxidation of this residue to methionine sulfoxide (MetO), which is reduced to methionine by the methionine sulfoxide reductase (Msr) system. However, under oxidative stress conditions other Met residues may also be oxidized, causing reduction in COMT’s activity.
Accordingly, we propose to overexpress recombinant Val108 and Met108 variants of soluble COMT in msrA null mutant and wild-type control bacterial cells, in the presence and absence of oxidative reagent. The resulting purified COMT variants will be crystalized and their structures will be determined. Our hypothesis is that COMT proteins containing MetO residues will exhibit altered structures and reduced activities that will be exacerbated in correlation with the presence of specific MetO residues. The acquired new data will lead to further investigations on how the resulting structural changes affect the binding of COMT to its substrate (catechol) and S-adenosyl methionine (SAMe). The subterrain Israeli blind mole rat (Spalax) contains a leucine (Leu) instead of a conserved Met in all other species, at amino-acid 187. This naturally occurring amino acid substitution may hint on the vulnerability of Met187 to oxidation.
We will be determining
Aim 1: The effect of Met/MetO residues on human and Spalax COMT’s enzymatic activity
Aim 2: The effect of Met/MetO residues on COMT’s protein structure.
Resulting information will shade light on the role of MetO residues on COMT’s function and may contribute to the development of novel treatments to COMT related diseases.