Dual role of the carboxyl-terminal region of pig liver L-kynurenine 3-monooxygenase: mitochondrial-targeting signal and enzymatic activity.
l-kynurenine 3-monooxygenase (KMO) is an NAD(P)H-dependent flavin monooxygenase that catalyses the hydroxylation of l-kynurenine to 3-hydroxykynurenine, and is localized as an oligomer in the mitochondrial outer membrane. In the human brain, KMO may play an important role in the formation of two neurotoxins, 3-hydroxykynurenine and quinolinic acid, both of which provoke severe neurodegenerative diseases. In mosquitos, it plays a role in the formation both of eye pigment and of an exflagellation-inducing factor (xanthurenic acid). Here, we present evidence that the C-terminal region of pig liver KMO plays a dual role. First, it is required for the enzymatic activity. Second, it functions as a mitochondrial targeting signal as seen in monoamine oxidase B (MAO B) or outer membrane cytochrome b(5). The first role was shown by the comparison of the enzymatic activity of two mutants (C-terminally FLAG-tagged KMO and carboxyl-terminal truncation form, KMOΔC50) with that of the wild-type enzyme expressed in COS-7 cells. The second role was demonstrated with fluorescence microscopy by the comparison of the intracellular localization of the wild-type, three carboxyl-terminal truncated forms (ΔC20, ΔC30 and ΔC50), C-terminally FLAG-tagged wild-type and a mutant KMO, where two arginine residues, Arg461-Arg462, were replaced with Ser residues.
Hirai
Kumiko
K
Neuronal Signaling Research Team, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo, Japan.
Kuroyanagi
Hidehito
H
Tatebayashi
Yoshitaka
Y
Hayashi
Yoshitaka
Y
Hirabayashi-Takahashi
Kanako
K
Saito
Kuniaki
K
Haga
Seiich
S
Uemura
Tomihiko
T
Izumi
Susumu
S
D000595:Amino Acid Sequence; D000818:Animals; D055162:Biocatalysis; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004789:Enzyme Activation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D007737:Kynurenine / Q000378:metabolism*; D050601:Kynurenine 3-Monooxygenase / Q000235:genetics / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology*; D051336:Mitochondrial Membranes / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D009842:Oligopeptides; D010455:Peptides; D017434:Protein Structure, Tertiary / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D017421:Sequence Analysis; D017384:Sequence Deletion; D013552:Swine
D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D009842:Oligopeptides; D010455:Peptides; D011994:Recombinant Proteins; D007737:Kynurenine; D009249:NADP; C085706:FLAG peptide; D050601:Kynurenine 3-Monooxygenase