cDNA cloning, expression and activity of a second human aflatoxin B1-metabolizing member of the aldo-keto reductase superfamily, AKR7A3.
The aflatoxin B1 (AFB1) aldehyde metabolite of AFB1 may contribute to the cytotoxicity of this hepatocarcinogen via protein adduction. Aflatoxin B1 aldehyde reductases, specifically the NADPH-dependent aldo-keto reductases of rat (AKR7A1) and human (AKR7A2), are known to metabolize the AFB1 dihydrodiol by forming AFB1 dialcohol. Using a rat AKR7A1 cDNA, we isolated and characterized a distinct aldo-keto reductase (AKR7A3) from an adult human liver cDNA library. The deduced amino acid sequence of AKR7A3 shares 80 and 88% identity with rat AKR7A1 and human AKR7A2, respectively. Recombinant rat AKR7A1 and human AKR7A3 were expressed and purified from Escherichia coli as hexa-histidine tagged fusion proteins. These proteins catalyzed the reduction of several model carbonyl-containing substrates. The NADPH-dependent formation of AFB1 dialcohol by recombinant human AKR7A3 was confirmed by liquid chromatography coupled to electrospray ionization mass spectrometry. Rabbit polyclonal antibodies produced using recombinant rat AKR7A1 protein were shown to detect nanogram amounts of rat and human AKR7A protein. The amount of AKR7A-related protein in hepatic cytosols of 1, 2-dithiole-3-thione-treated rats was 18-fold greater than in cytosols from untreated animals. These antibodies detected AKR7A-related protein in normal human liver samples ranging from 0.3 to 0.8 microg/mg cytosolic protein. Northern blot analysis showed varying levels of expression of AKR7A RNA in human liver and in several extrahepatic tissues, with relatively high levels in the stomach, pancreas, kidney and liver. Based on the kinetic parameters determined using recombinant human AKR7A3 and AFB1 dihydrodiol at pH 7.4, the catalytic efficiency of this reaction (k2/K, per M/s) equals or exceeds those reported for other enzymes, for example cytochrome P450s and glutathione S-transferases, known to metabolize AFB1 in vivo. These findings indicate that, depending on the extent of AFB1 dihydrodiol formation, AKR7A may contribute to the protection against AFB1-induced hepatotoxicity.
Knight
L P
LP
Department of Environmental Health Sciences, Johns Hopkins School of Hygiene and Public Health, Baltimore, MD 21205, USA.
Primiano
T
T
Groopman
J D
JD
Kensler
T W
TW
Sutter
T R
TR
D016604:Aflatoxin B1 / Q000031:analogs & derivatives / Q000378:metabolism*; D000449:Aldehyde Reductase / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000074408:Aldo-Keto Reductases; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001547:Benzaldehydes / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics; D010616:Phenanthrenes / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
D001547:Benzaldehydes; D018076:DNA, Complementary; D010616:Phenanthrenes; D012333:RNA, Messenger; C005399:9,10-phenanthrenequinone; C029915:4-nitrobenzaldehyde; D016604:Aflatoxin B1; D000074408:Aldo-Keto Reductases; C000603089:AKR7A3 protein, human; D000449:Aldehyde Reductase