Inducible (1 R,2S)-1,2-dihydroxy-3,5-cyclohexadiene-l,4-dicarboxylate (diene-dial) dehydrogenase 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, 39000. It had a specific activity of 500 mkat/kg of protein and was activated by the addition of Fe". The dehydrogenase converted 1 mol diene-diol and 1 mol NAD+ to 1 mol protocatechuic acid, 1 mol NADH and 1 mol CO,. Apparent Km-values of 43 PM (NAD+C) and about 90 PM (diene-dial) were determined. The hydride ion was transferred to the si face of NAD+.
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