Carbonyl reductase sniffer from the model organism daphnia: Cloning, substrate determination and inhibitory sensitivity

Carbonyl reductases (CRs) represent a fundamental enzymatic defense mechanism against oxidative stress. While commonly two carbonyl reductases (CBR1 and CBR3) are found in mammalian genomes, invertebrate model organisms like Drosophila melanogaster express no CR but a functional homolog to human CBR1, termed sniffer. The importance of sniffer could be demonstrated in D. melanogaster where it protected against age-dependent neurodegeneration. Interestingly, the microcrustacean Daphnia harbors four copies of the CR gene (CR1, CR2, CR3, CR4) in addition to one sniffer gene. Due to this unique equipment Daphnia is an ideal model organism to investigate the function of sniffer. Recombinant sniffer from D. magna und D. pules were produced in E. coli, purified by Ni-affinity chromatography and tested with a variety of aliphatic and aromatic diketones, reactive aldehydes and precursors of advanced glycation end products (AGE). The highest catalytic activities were determined for sniffer from D. pulex with the aromatic dicarbonyls 9,10-phenanthrenequinone (k_cat/K_m = 2.6 s^-1 x μM^-1) and isatin (k_cat/K_m = 1.5 s^-1 x μM^-1). While sniffer from D. magna displayed preference for the same two substances, the respective catalytic activities were noticeably lower. Kinetic constants with aliphatic diketones were generally lower than those with aromatic dicarbonyls for both sniffer enzymes. The best aliphatic diketone as substrate for sniffer from D. magna and D. pulex was hexane-3,4-dione with k_cat/K_m = 0.23 s^-1 μM^-1 and k_cat/K_m = 0.35 s^-1 μM^-1, respectively. Poor or no detectable activity of the two sniffer enzymes was seen with the aliphatic diketones 2,5-hexanedione and 3,5-heptanedione, the aldehydes butanal, hexanal, decanal, crotonaldehyde, acrolein, trans-2-hexenal, and the AGE precursors glyoxal, methylglyoxal, furfural and glyceraldehyde, indicating no physiological function in the metabolism of short-chain aldehydes. Substrate inhibition for both sniffer enzymes was observed with the quinone substrates 1,4-naphthoquinone and 2-methyl-1,4-benzoquinone. From a variety of pesticides endosulfan turned out as an effective inhibitor of the sniffer enzymes (Ki = 9.2 μM for sniffer from D. magna, Ki = 12.0 μM for sniffer from D. pulex). In conclusion, the present results on sniffer from the protein superfamily of the short-chain dehydrogenases/reductases (SDR) in Daphnia ssp. complement earlier studies on carbonyl reductases in the same species and indicate that Daphnia is an interesting model to study the overall response to carbonyl stress.