PURIFICATION AND SOME PROPERTIES OF 1,2-DIHYDROXY-3,5-CYCLOHEXADIENE-1,4-DICARBOXYLATE DEHYDROGENASE FROM COMAMONAS-TESTOSTERONI T-2

Inducible (1R,2S)-1,2-dihydroxy-3,5-cyclohexadiene (diene-diol) dehydr ogenase was found in extracts of Comamonas testosteroni T-2 grown in p -toluate-or terephthalate-salts medium and it was purified using anion exchange, hydrophobic interaction and gel filtration chromatography. The enzyme is a homodimer with subunit M(r) 39 000. It had a specific activity of 500 mkat/kg of protein and was activated by the addition o f Fe2+. The dehydrogenase converted 1 mol diene-diol and 1 mol NAD(+) to 1 mol protocatechuic acid, 1 mol NADH and 1 mol CO2. Apparent K-m-v alues of 43 mu M (NAD(+)) and about 90 mu M (diene-diol) were determin ed. The hydride ion was transferred to the si face of NAD(+).