Transhydroxylase of Pelobacter acidigallici: a molybdoenzyme catalyzing the conversion of pyrogallol to phloroglucinol.
Trihydroxybenzenes are degraded anaerobically through the phloroglucinol pathway. In Pelobacter acidigallici as well as in Pelobacter massiliensis, pyrogallol is converted to phloroglucinol in the presence of 1,2,3,5-tetrahydroxybenzene by intermolecular hydroxyl transfer. The enzyme catalyzing this reaction was purified to chromatographic and electrophoretic 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 subunits. The enzyme was not sensitive to oxygen. HgCl2, p-chloromercuribenzoic acid, and CuCl2 inhibited strongly the reaction indicating an essential 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 tetrahydroxybenzene was 31.4 kJ per mol. Purified enzyme exhibited a specific activity of 3.1 mol min-1 mg-1 protein and an apparent Km for 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. Requirement 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.
Reichenbecher
W
W
Fakultät für Biologie, Universität Konstanz, FRG.
Brune
A
A
Schink
B
B
D001421:Bacteria, Anaerobic / Q000378:metabolism*; D004795:Enzyme Stability; D007501:Iron / Q000032:analysis; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008982:Molybdenum / Q000032:analysis; D010084:Oxidation-Reduction; D010696:Phloroglucinol / Q000378:metabolism*; D011748:Pyrogallol / Q000378:metabolism*; D013455:Sulfur / Q000032:analysis
D011748:Pyrogallol; D013455:Sulfur; D008982:Molybdenum; D010696:Phloroglucinol; D007501:Iron; D006899:Mixed Function Oxygenases