Towards the reaction mechanism of pyrogallol-phloroglucinol transhydroxylase of Pelobacter acidigallici

Conversion of pyrogallol to phloroglucinol was studied with the molybdenum enzyme transhydroxylase of the strictly anaerobic fermenting bacterium Pelo bacter acidigallici. Transhydroxylation experiments in (H2O)-O-18 revealed that none of the hydroxyl groups of phloroglucinol was derived from water, confirming the concept that this enzyme transfers a hydroxyl group, from th e cosubstrate 1,2,3,5-tetrahydroxybenzene (tetrahydroxybenzene) to the acce ptor pyrogallol, and simultaneously regenerates the cosubstrate. This conce pt requires a reaction which synthesizes the cofactor de novo to maintain a sufficiently high intracellular pool during growth. Some sulfoxides and ar omatic N-oxides were found to act as hydroxyl donors to convert pyrogallol to tetrahydroxybenzene. Again, water was not the source of the added hydrox yl groups; the oxides reacted as cosubstrates in a transhydroxylation react ion rather than as true oxidants in a net hydroxylation reaction. No oxidiz ing agent was found that supported a formation of tetrahydroxybenzene via a net hydroxylation of pyrogallol. However, conversion of pyrogallol to phlo roglucinol in the absence of tetrahydroxybenzene was achieved if little pyr ogallol and a high amount of enzyme preparation was used which had been pre -exposed to air. Obviously, the enzyme was oxidized by air to form sufficie nt amounts of tetrahydroxybenzene from pyrogallol to start the reaction. A reaction mechanism is proposed which combines an oxidative hydroxylation wi th a reductive dehydroxylation via the molybdenum cofactor, and allows the transfer of a hydroxyl group between tetrahydroxybenzene and pyrogallol wit hout involvement of water. With this, the transhydroxylase differs basicall y from all other hydroxylating molybdenum enzymes which all use water as hy droxyl source. (C) 1999 Elsevier Science B.V. All rights reserved.