Molecular cloning of human D-dopachrome tautomerase cDNA: N-terminal proline is essential for enzyme activation.

D-Dopachrome tautomerase converts 2-carboxy-2,3-dihydroindole-5,6-quinone (D-dopachrome) into 5,6-dihydroxyindole. This protein has an amino acid sequence that is highly homologous with that of macrophage migration inhibitory factor (MIF), which has the potential to catalyze D-dopachrome to 5,6-dihydroxyindole-2-carboxylic acid and is an important cytokine for T-lymphocyte activation. We isolated and sequenced a 566 bp-long cDNA encoding human D-dopachrome tautomerase. The cDNA contains an open reading frame encoding 118 amino acids, including the initiator methionine. The amino acid sequence of the protein shares 80% homology with that of the rat enzyme. Northern blot analysis demonstrated that mRNA of D-dopachrome tautomerase is expressed in a large amount in the liver, and to lesser extent in other organs, including the heart, lung and pancreas. After purification of D-dopachrome tautomerase expressed in E. coli, we confirmed that the recombinant protein catalyzes the conversion of D-dopachrome to 5,6-dihydroxyindole. Its catalytic mechanism is not well understood. We found that the protein completely lost the enzyme activity when the N-terminal proline residue was replaced with alanine by site-directed mutagenesis. This fact suggests that the N-terminal proline is essential for the catalytic mechanism. Although the precise pathophysiological function of D-dopachrome tautomerase remains to be elucidated, the present results could contribute to further understanding of isomerase activity in relation to the immune response.