DOI: 10.1002/chem.200601053 Investigation of the Mechanism of Action of Pyrogallol–Phloroglucinol Transhydroxylase by Using Putative Intermediates Csaba Paizs, [a, b] Ulrike Bartlewski-Hof, [a] and Jµnos RØtey* [a] Dedicated to Professor Bernhard Kräutler on the occasion of his 60th birthday Introduction Pyrogallol–phloroglucinol transhydroxylase (TH) from Pelo- bacter acidigallici is one of the key enzymes in the anaerobic degradation of aromatic compounds like gallic acid and vari- ous phenols. [1–3] All these compounds are converted into phloroglucinol, which is then reductively dearomatized and degraded to three acetyl-coenzyme A (acetyl-CoA) mole- cules via 3-hydroxy-5-oxohexanoate. This pathway leads not only to important building blocks but can also be used for adenosine triphosphate (ATP) synthesis. Schink and co-workers showed that TH contains a molyb- dopterin cofactor (Moco) and iron sulphur clusters [4Fe–4S] and needs 1,2,3,5-tetrahydroxybenzene as a cosubstrate, or rather as a cocatalyst because it is regenerated in the reac- tion cycle. [1–6] Furthermore, as shown by 18 O labeling, no hy- droxy groups are incorporated into the product from water. [5] On the basis of these results, the reaction was for- mulated [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 cocata- lyst are produced. Such ingenious use of a cocatalyst already has precedence in enzyme chemistry; for example, the gly- colytic enzymes, phosphoglucomutase and phosphoglycerate mutase, take advantage of their cocatalysts, that is, glucose- 1,6-bisphosphate and glycerate-2,3-bisphosphate, respective- Abstract: Pyrogallol–phloroglucinol transhydroxylase from Pelobacter acidi- gallici, a molybdopterin-containing enzyme, catalyzes a key reaction in the anaerobic degradation of aromatic compounds. In vitro, the enzymatic re- action requires 1,2,3,5-tetrahydroxy- benzene as a cocatalyst and the trans- hydroxylation occurs without exchange with hydroxy groups from water. To test our previous proposal that the transfer of the hydroxy group occurs via 2,4,6,3’,4’,5’-hexahydroxydiphenyl ether as an intermediate, we synthe- sized this compound and investigated its properties. We also describe the syn- thesis and characterization of 3,4,5,3’,4’,5’-hexahydroxydiphenyl ether. Both compounds could substi- tute for the cocatalyst in vitro. This in- dicates that the diphenyl ethers can in- trude into the active site and initiate the catalytic cycle. Recently, the X-ray crystal structure of the transhydroxy- lase (TH) was published [16] and it sup- ports the proposed mechanism of hy- droxy-group transfer. Keywords: enzymes · ethers · molybdopterin (Moco) · OH transfer · reaction mechanisms [a] Dr. Cs. Paizs, U. Bartlewski-Hof, Prof. Dr. J. RØtey Institute of Organic Chemistry and Biochemistry University of Karlsruhe Richard-Willstätter-Allee, 76128 Karlsruhe (Germany) Fax: (+ 49) 721-608-4823 E-mail: janos.retey@ioc.uka.de [b] Dr. Cs. Paizs Department of Biochemistry and Biochemical Engineering Babes-Bolyai University Arany Jµnos str. 11, 400028 Cluj-Napoca (Kolozsvµr) (Romania) Scheme 1. The role of 1,2,3,5-tetrahydroxybenzene in the mechanism of action of the Mo-cofactor-containing pyrogallol–phloroglucinol transhy- droxylase as postulated by Schink and co-workers. [4, 5] Chem. Eur. J. 2007, 13, 2805 – 2811 # 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 2805 FULL PAPER