Cloning of human 3-hydroxyanthranilic acid dioxygenase in Escherichia coli : characterisation of the puri¢ed enzyme and its in vitro inhibition by Zn 2þ Vito Calderone a ;  , Michela Trabucco a , Valentina Menin a , Alessandro Negro b , Giuseppe Zanotti a a Department of Organic Chemistry and Biopolymer Research Center, University of Padua, via Marzolo 1, 35131 Padua, Italy b CRIBI, University of Padua, via Ugo Bassi 58/B, 35131 Padua, Italy Received 27 April 2001; received in revised form 19 December 2001; accepted 10 January 2002 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, Zn 2þ , could be of physiological relevance. A circular dichroism analysis has also been performed, showing that the secondary structure is mainly of the L type, with a minority of K. ß 2002 Elsevier Science B.V. All rights reserved. Keywords : 3-Hydroxyanthranilic acid oxygenase ; Zinc ; Quinolinic acid ; Neurodegenerative disease 1. Introduction In mammalian peripheral organs, 3-hydroxyan- thranilic acid dioxygenase (3-HAO), catalysing the conversion of 3-hydroxyanthranilic acid (3-HANA) to quinolinic acid (2,3-pyridinedicarboxylic acid; QUIN), constitutes a link in the catabolic pathway of tryptophan to NAD (Fig. 1). QUIN is a naturally occurring acid and a structural analogue of neuro- transmitters like L-glutamate and L-aspartate. When introduced directly into the mammalian central ner- vous system (CNS), QUIN is a potent excitotoxin that acting through excitatory amino acid receptors causes neuronal excitation [1], seizure activity [2] and neuronal degeneration [3]. More precisely, it has also been suggested that increased cerebral levels of QUIN may participate in the pathogenesis of neuro- logical disorders such as epilepsy [4], Huntington disease [5,6] and damages deriving from brain trau- ma and hypoxic states [7]. Of considerable interest is the proposed role of QUIN as mediator for neuro- degeneration occurring in in£ammatory conditions [8,9]. In fact, increased and possibly neurotoxic levels of QUIN have been detected in the cerebrospinal £uid and serum of humans with infectious diseases (e.g. in patients a¡ected by AIDS and Lyme borre- 0167-4838 / 02 / $ ^ see front matter ß 2002 Elsevier Science B.V. All rights reserved. PII:S0167-4838(02)00216-9 * Corresponding author. Fax : +39-09-827-5239. E-mail address : vito@chor.unipd.it (V. Calderone). Biochimica et Biophysica Acta 1596 (2002) 283^292 www.bba-direct.com