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

Abstract

Conversion of pyrogallol to phloroglucinol was studied with the molybdenum enzyme transhydroxylase of the strictly anaerobic fermenting bacterium Pelobacter acidigallici. Transhydroxylation experiments in H 2 18 O revealed that none of the hydroxyl groups of phloroglucinol was derived from water, confirming the concept that this enzyme transfers a hydroxyl group from the cosubstrate 1,2,3,5-tetrahydroxybenzene (tetrahydroxybenzene) to the acceptor pyrogallol, and simultaneously regenerates the cosubstrate. This concept requires a reaction which synthesizes the cofactor de novo to maintain a sufficiently high intracellular pool during growth. Some sulfoxides and aromatic N-oxides were found to act as hydroxyl donors to convert pyrogallol to tetrahydroxybenzene. Again, water was not the source of the added hydroxyl groups; the oxides reacted as cosubstrates in a transhydroxylation reaction rather than as true oxidants in a net hydroxylation reaction. No oxidizing agent was found that supported a formation of tetrahydroxybenzene via a net hydroxylation of pyrogallol. However, conversion of pyrogallol to phloroglucinol in the absence of tetrahydroxybenzene was achieved if little pyrogallol 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 sufficient amounts of tetrahydroxybenzene from pyrogallol to start the reaction. A reaction mechanism is proposed which combines an oxidative hydroxylation with a reductive dehydroxylation via the molybdenum cofactor, and allows the transfer of a hydroxyl group between tetrahydroxybenzene and pyrogallol without involvement of water. With this, the transhydroxylase differs basically from all other hydroxylating molybdenum enzymes which all use water as hydroxyl source.

The Mononuclear Molybdenum Enzymes

“…The hydroxyl group that is transferred is not derived from solvent, nor does it equilibrate with solvent in the course of turnover. 532 The crystal structure of the transhydroxylase (Figure 59) shows the enzyme to be an αβ heterodimer, with a molybdenum-containing large subunit and a smaller subunit with three [4Fe-4S] clusters. 533 The large subunit consists of four domains as seen in the Rhodobacter DMSO reductases (Figure 43), although segments of the chain trace (mostly in regions involved in substrate binding) differ significantly from that seen in DMSO reductase.…”

Identification and characterization of hydroxyquinone hydratase activities from Sphingobium chlorophenolicum ATCC 39723

“…Inside cells, 1,2,4,5-tetrahydroxybenezene may be further metabolized without the formation of DHBQ, especially under anaerobic conditions. Tetrahydroxybenzene can be used in transhydroxylation reactions with hydroxyquinol, pyrogallol and phloroglucinol, or cleaved by a ring-cleavage dioxygenase, as previously reported for the metabolism of related compounds Haddock & Ferry 1993;Krumholz & Bryant 1988;Reichenbecher & Schink 1999). Thus, the presence of a hydroxyquinone hydratase in S. chlorophenolicum indicates the possible presence of an as yet undiscovered aromatic catabolism pathway in this organism.…”

Investigation of the Mechanism of Action of Pyrogallol–Phloroglucinol Transhydroxylase by Using Putative Intermediates

“…When pure transhydroxylase, isolated as described by Schink and co-workers, [5] was incubated with pyrogallol only, no reaction occurred as monitored by HPLC. In the presence of 1, phloroglucinol was produced; after a longer incubation (15 min) at 30 8C, the concentration of 1 was only slightly decreased (Figure 2), as it was regenerated in the reaction cycle.…”

“…The enzyme assay is a modification of that described by Schink and co-workers. [4][5][6] Based on the areas under the HPLC traces and the amounts of the pyrogallol (10 mmol) and enzyme (0.1 mg) used in the reaction, the enzyme activity can be estimated to be 2 mmol min À1 mg…”

“…[1][2][3][4][5][6] Furthermore, as shown by 18 O labeling, no hydroxy groups are incorporated into the product from water. [5] On the basis of these results, the reaction was formulated [4,5] as depicted in Scheme 1. The 2-OH group from 1,2,3,5-tetrahydroxybenzene is transferred to pyrogallol, whereby phloroglucinol and a new molecule of the cocatalyst are produced.…”