Mouse liver NAD(P)H:quinone acceptor oxidoreductase: Protein sequence analysis by tandem mass spectrometry, cDNA cloning, expression in Escherichia coli, and enzyme activity analysis

Abstract

The amino acid sequence of mouse liver NAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2) has been determined by tandem mass spectrometry and deduced from the nucleotide sequence of the cDNA encoding for the enzyme. The electrospray mass spectral analyses revealed, as previously reported (Prochaska HJ, Talalay P, 1986, J Biol Chem 261:1372–1378), that the 2 forms – the hydrophilic and hydrophobic forms – of the mouse liver quinone reductase have the same molecular weight. No amino acid sequence differences were found by tandem mass spectral analyses of tryptic peptides of the 2 forms. Moreover, the amino‐termini of the mouse enzymes are acetylated as determined by tandem mass spectrometry. Further, only 1 cDNA species encoding for the quinone reductase was found. These results suggest that the 2 forms of the mouse quinone reductase have the same primary sequences, and that any difference between the 2 forms may be attributed to a labile posttranslational modification.Analysis of the mouse quinone reductase cDNA revealed that the enzyme is 273 amino acids long and has a sequence homologous to those of rat and human quinone reductases. In this study, the mouse quinone reductase cDNA was also ligated into a prokaryotic expression plasmid pKK233.2, and the constructed plasmid was used to transform Escherichia coli strain JM109. The E. coli‐expressed mouse quinone reductase was purified and characterized. Although mouse quinone reductase has an amino acid sequence similar to those of the rat and human enzymes, the mouse enzyme has a higher NAD(P)H‐menadione reductase activity and is less sensitive to flavones and dicoumarol, 2 known inhibitors of the enzyme. The results would indicate that the regions in mouse quinone reductase that contain amino acids different from the rat and human enzymes are critical for the binding of menadione, flavones, and dicoumarol.