TRANSHYDROXYLASE OF PELOBACTER-ACIDIGALLICI - A MOLYBDOENZYME CATALYZING THE CONVERSION OF PYROGALLOL TO PHLOROGLUCINOL

Trihydroxybenzenes are degraded anaerobically through the phloroglucin ol pathway. In Pelobacter acidigallici as well as in Pelobacter massil iensis, pyrogallol is converted to phloroglucinol in the presence of 1 ,2,3,5-tetrahydroxybenzene by intermolecular hydroxyl transfer. The en zyme catalyzing this reaction was purified to chromatographic and elec trophoretic homogeneity. Gel filtration and electrophoresis revealed a heterodimer structure with an apparent molecular mass of 127 kDa for the native enzyme and 86 kDa and 38 kDa, respectively, for the subunit s. The enzyme was not sensitive to oxygen. HgCl2, p-chloromercuri-benz oic acid, and CuCl2 inhibited strongly the reaction indicating an esse ntial function of SH-groups. Transhydroxylase had a pH-optimum of 7.0 and a pI of 4.1. The apparent temperature optimum was in the range of 53 degrees C to 58 degrees C. The activation energy for the conversion of pyrogallol and 1,2,3,5-tetrahydroxybenzene to phloroglucinol and t etrahydroxybenzene was 31.4 kJ per mel. Purified enzyme exhibited a sp ecific activity of 3.1 mol min(-1) mg(-1)protein and an apparent K-m f or pyrogallol and 1,2,3,5-tetrahydroxybenzene of 0.70 mM and 0.71 mM, respectively. The enzyme was found to contain per mol heterodimer 1.1 mol molybdenum, 12.1 mol iron and 14.5 mol acid-labile sulfur. Require ment for molybdenum for transhydroxylating enzyme activity was proven also by cultivation experiments. No hints for the presence of flavins were obtained. The results presented here support the hypothesis that a redox reaction is involved in this intermolecular hydroxyl transfer.