A single amino acid substitution confers high cinchonidine oxidation activity comparable with that of rabbit to monkey aldehyde oxidase 1.
Aldehyde oxidase 1 (AOX1) is a major member of the xanthine oxidase family belonging to the class of complex molybdo-flavoenzymes and plays an important role in the nucleophilic oxidation of N-heterocyclic aromatic compounds and various aldehydes. The enzyme has been well known to show remarkable species differences. Comparing the rabbit and monkey enzymes, the former showed extremely high activity toward cinchonidine and methotrexate, but the latter exhibited only marginal activities. In contrast, monkey had several times greater activity than did rabbit toward zonisamide and (+)-4-(4-cyanoanilino)-5,6-dihydro-7-hydroxy-7H-cyclopenta[d]-pyrimidine [(S)-RS-8359]. In this report, we tried to confer high cinchonidine oxidation activity comparable with that of rabbit AOX1 to monkey AOX1. The chimera proteins prepared by restriction enzyme digestion and recombination methods between monkey and rabbit AOX1s indicated that the sequences from Asn993 to Ala1088 of rabbit AOX1 are essential for the activity. The kinetic parameters were then measured using monkey AOX1 mutants prepared by site-directed mutagenesis. The monkey V1085A mutant acquired the high cinchonidine oxidation activity. Inversely, the reciprocal rabbit A1081V mutant lost the activity entirely: amino acid 1081 of rabbit AOX1 corresponding to amino acid 1085 of monkey AOX1. Thus, cinchonidine oxidation activity was drastically changed by mutation of a single residue in AOX1. However, this might be true for bulky substrates such as cinchonidine but not for small substrates. The mechanism of substrate-dependent species differences in AOX1 activity toward bulky substrates is discussed.
Fukiya
Kensuke
K
Department of Drug Metabolism and Pharmacokinetics, Tohoku Pharmaceutical University, Sendai, Japan.
Itoh
Kunio
K
Yamaguchi
Satoshi
S
Kishiba
Akiko
A
Adachi
Mayuko
M
Watanabe
Nobuaki
N
Tanaka
Yorihisa
Y
D042931:Aldehyde Oxidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D020134:Catalytic Domain; D002930:Cinchona Alkaloids / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008252:Macaca fascicularis; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009570:Nitriles / Q000378:metabolism; D010084:Oxidation-Reduction; D010957:Plasmids; D011743:Pyrimidines / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013045:Species Specificity; D013379:Substrate Specificity
D002930:Cinchona Alkaloids; D007527:Isoenzymes; D050505:Mutant Proteins; D009570:Nitriles; D011743:Pyrimidines; D011993:Recombinant Fusion Proteins; C041622:cinchonidine; C059590:RS 8359; D042931:Aldehyde Oxidase