Biochemical and structural properties of mouse kynurenine aminotransferase III.
Kynurenine aminotransferase III (KAT III) has been considered to be involved in the production of mammalian brain kynurenic acid (KYNA), which plays an important role in protecting neurons from overstimulation by excitatory neurotransmitters. The enzyme was identified based on its high sequence identity with mammalian KAT I, but its activity toward kynurenine and its structural characteristics have not been established. In this study, the biochemical and structural properties of mouse KAT III (mKAT III) were determined. Specifically, mKAT III cDNA was amplified from a mouse brain cDNA library, and its recombinant protein was expressed in an insect cell protein expression system. We established that mKAT III is able to efficiently catalyze the transamination of kynurenine to KYNA and has optimum activity at relatively basic conditions of around pH 9.0 and at relatively high temperatures of 50 to 60 degrees C. In addition, mKAT III is active toward a number of other amino acids. Its activity toward kynurenine is significantly decreased in the presence of methionine, histidine, glutamine, leucine, cysteine, and 3-hydroxykynurenine. Through macromolecular crystallography, we determined the mKAT III crystal structure and its structures in complex with kynurenine and glutamine. Structural analysis revealed the overall architecture of mKAT III and its cofactor binding site and active center residues. This is the first report concerning the biochemical characteristics and crystal structures of KAT III enzymes and provides a basis toward understanding the overall physiological role of mammalian KAT III in vivo and insight into regulating the levels of endogenous KYNA through modulation of the enzyme in the mouse brain.
Han
Qian
Q
Department of Biochemistry, Virginia Tech, Blacksburg, Virginia 24061, USA.
Robinson
Howard
H
Cai
Tao
T
Tagle
Danilo A
DA
Li
Jianyong
J
D000586:Amination; D000818:Animals; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005973:Glutamine / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007737:Kynurenine / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature; D000637:Transaminases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D021122:Protein Subunits; D005973:Glutamine; D007737:Kynurenine; D011732:Pyridoxal Phosphate; D000637:Transaminases; C021481:kynurenine-oxoglutarate transaminase