Abstract
Kynurenine aminotransferase from Pyrococcus horikoshii OT3 (PhKAT), which is a homodimeric protein, catalyzes the conversion of kynurenine (KYN) to kynurenic acid (KYNA). We analyzed the transaminase reaction mechanisms of this protein with pyridoxal-5′-phosphate (PLP), KYN and α-ketoglutaric acid (2OG) or oxaloacetic acid (OXA). 2OG significantly inhibited KAT activities in kinetic analyses, suggesting that a KYNA biosynthesis is allosterically regulated by 2OG. Its inhibitions evidently were unlocked by KYN. 2OG and KYN functioned as an inhibitor and activator in response to changes in the concentrations of KYN and 2OG, respectively. The affinities of one subunit for PLP or 2OG were different from that of the other subunit, as confirmed by spectrophotometry and isothermal titration calorimetry, suggesting that the difference of affinities between subunits might play a role in regulations of the KAT reaction. Moreover, we identified two active and allosteric sites in the crystal structure of PhKAT-2OG complexes. The crystal structure of PhKAT in complex with four 2OGs demonstrates that two 2OGs in allosteric sites are effector molecules which inhibit the KYNA productions. Thus, the combined data lead to the conclusion that PhKAT probably is regulated by allosteric control machineries, with 2OG as the allosteric inhibitor.
Highlights
Kynurenine-oxoglutarate transaminase (EC 2.6.1.7), known as kynurenine (KYN) aminotransferase (KAT) [1], is a dimeric enzyme containing 2 covalently-bound pyridoxal-59phosphates (PLP) as cofactor moieties and is the last enzyme in the kynurenic acid (KYNA) biosynthetic pathway
PhKAT (48 kDa), which was expressed as a soluble protein, was detected by sodium dodecyl sulfate (SDS)-PAGE (Fig. 1A)
The results of this study show that the mechanisms of KYNA synthesis from KYN is conserved in the hyperthermophilic archaeon, P. horikoshii, and that KYN transamination results in the production of KYNA (Figs. 3 and 4)
Summary
Kynurenine-oxoglutarate transaminase (EC 2.6.1.7), known as kynurenine (KYN) aminotransferase (KAT) [1], is a dimeric enzyme containing 2 covalently-bound pyridoxal-59phosphates (PLP) as cofactor moieties and is the last enzyme in the kynurenic acid (KYNA) biosynthetic pathway. Kynurenic acid (KYNA) is biosynthesized as a product of the normal metabolism of the amino acid L-tryptophan and via a KYN intermediate; KYNA is synthesized via the transamination of KYN in the presence of KAT. KYNA is sequentially biosynthesized from LKYN via a 4-(2-aminophenyl)-2,4-dioxobutanoate (4AD) intermediate by KAT (Fig. S1). KAT transfers the amino group of KYN to a-ketoglutaric acid (2OG) via pyridoxamine phosphate (PMP), synthesizing L-glutamic acid (Glu). KYNA acts as a natural antagonist of the glycine site of NMDA (N-methyl D-aspartate) receptor (NMDAR) and plays a key role in the glutamatergic neurotransmission system [2]. The KAT from the hyperthermophilic archaeon, Pyrococcus horikoshii OT3 (PhKAT), is a homolog of human KAT II
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