Mitochondrial aspartate aminotransferase: a third kynurenate-producing enzyme in the mammalian brain Paolo Guidetti, Laura Amori, Michael T. Sapko, Etsuo Okuno 1 and Robert Schwarcz Maryland Psychiatric Research Center, University of Maryland School of Medicine, Baltimore, Maryland, USA Abstract The tryptophan metabolite kynurenic acid (KYNA), which is produced enzymatically by the irreversible transamination of L-kynurenine, is an antagonist of a7 nicotinic and NMDA re- ceptors and may thus modulate cholinergic and glutamatergic neurotransmission. Two kynurenine aminotransferases (KAT I and II) are currently considered the major biosynthetic en- zymes of KYNA in the brain. In this study, we report the existence of a third enzyme displaying KAT activity in the mammalian brain. The novel KAT had a pH optimum of 8.0 and a low capacity to transaminate glutamine or a-aminoadi- pate (the classic substrates of KAT I and KAT II, respectively). The enzyme was inhibited by aspartate, glutamate, and quisqualate but was insensitive to blockade by glutamine or anti-KAT II antibodies. After purification to homogeneity, the protein was sequenced and the enzyme was identified as mitochondrial aspartate aminotransferase (mitAAT). Finally, the relative contributions of KAT I, KAT II, and mitAAT to total KAT activity were determined in mouse, rat, and human brain at physiological pH using anti-mitAAT antibodies. KAT II was most abundant in rat and human brain, while mitAAT played the major role in mouse brain. It remains to be seen if mitAAT participates in cerebral KYNA synthesis under physiological and/or pathological conditions in vivo. Keywords: excitotoxicity, kynurenine, neurodegeneration, NMDA, schizophrenia, a7 nicotinic. J. Neurochem. (2007) 102, 103–111. Kynurenic acid (KYNA), a product of the kynurenine pathway of tryptophan degradation, has recently attracted the attention of neuroscientists because of its ability to inhibit the a7 nicotinic acetylcholine receptor and the glycine co- agonist site of the NMDA receptor at low concentrations (Schwarcz and Pellicciari 2002). These receptors are Ca 2+ - permeable ion channels, which are critically involved in brain physiology, and their malfunction has been associated with numerous brain diseases including neurodegenerative disorders, drug addiction, and schizophrenia (Dajas-Bailador et al. 2000; Buisson and Bertrand 2002; Kelley 2004; Coyle 2006; Mazurov et al. 2006). Fluctuations in brain KYNA levels, which occur under physiological and pathological conditions, affect both of these receptors and may therefore play a role in the modulation of cholinergic and glutamat- ergic neurotransmission (Schwarcz and Pellicciari 2002). The mechanisms governing the formation and disposition of KYNA in the brain have been explored in several laboratories but are not yet fully understood. As in the periphery, brain KYNA is produced enzymatically by the irreversible transamination of L-kynurenine, the product of the oxidative opening of tryptophan’s indole ring. Newly produced KYNA readily enters the extracellular compart- ment (Turski et al. 1989; Speciale et al. 1990; Swartz et al. 1990). Subsequently, passive efflux, rather than further enzymatic degradation or re-uptake, is primarily responsible for its removal from the brain (Moroni et al. 1988; Turski and Schwarcz 1988). Cerebral KYNA function therefore appears to be regulated mainly by the availability of kynurenine and the activity of its biosynthetic enzymes. Two kynurenine aminotransferases (KATs) are currently believed to be responsible for KYNA formation in the mammalian brain. These enzymes, KAT I [glutamine transaminase K (GTK; EC 2.6.1.64)] and KAT II [a-aminoadipate aminotransferase (a-ADA; EC 2.6.1.7)], Received August 26, 2006; revised manuscript received December 22, 2006; accepted January 2, 2007. Address correspondence and reprint requests to Robert Schwarcz PhD, Maryland Psychiatric Research Center, PO Box 21247, Baltimore, MD 21228, USA. E-mail: rschwarc@mprc.umaryland.edu 1 The present address of Etsuo Okuno is the Kyushu Nutrition Welfare University, Faculty of Nutrition, Department of Clinical Nutrition, Kitakyushu, Fukuoka 803-8511, Japan. Abbreviations used: GTK, glutamine transaminase K; KAT, kynure- nine aminotransferase; KYNA, kynurenic acid; mitAAT, mitochondrial aspartate aminotransferase; a-ADA, a-aminoadipate aminotransferase. Journal of Neurochemistry , 2007, 102, 103–111 doi:10.1111/j.1471-4159.2007.04556.x Ó 2007 The Authors Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 103–111 103