Purification and some properties of (1 R,2 S)-1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate dehydrogenase from Comamonas testosteroni T-2

Abstract

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