Cloning of human 3-hydroxyanthranilic acid dioxygenase in Escherichia coli: characterisation of the purified enzyme and its in vitro inhibition by Zn2+

Abstract

3-hydroxyanthranilic acid oxygenase (3-HAO) catalyses the conversion of 3-hydroxyanthranilic acid to quinolinic acid. Because of the involvement of quinolinic acid in the initiation of neurodegenerative phenomena, we have cloned human 3-HAO in Escherichia coli, overexpressed and purified it with the aim of studying its enzymatic activity and for future structural studies. The recombinant human protein, obtained in E. coli, retains its enzymatic activity which can occur only in the presence of Fe(II); several other metals have been tested but in no case the formation of the product has been observed. On the contrary, two of the ions tested inhibit the catalytic reaction and one of them, Zn2+, could be of physiological relevance. A circular dichroism analysis has also been performed, showing that the secondary structure is mainly of the beta type, with a minority of alpha.