Crystal structure of pyrogallol–phloroglucinol transhydroxylase, an Mo enzyme capable of intermolecular hydroxyl transfer between phenols

Abstract

The Mo enzyme transhydroxylase from the anaerobic microorganism Pelobacter acidigallici catalyzes the conversion of pyrogallol to phloroglucinol. Such trihydroxybenzenes and their derivatives represent important building blocks of plant polymers. None of the transferred hydroxyl groups originates from water during transhydroxylation; instead a cosubstrate, such as 1,2,3,5-tetrahydroxybenzene, is used in a reaction without apparent electron transfer. Here, we report on the crystal structure of the enzyme in the reduced Mo(IV) state, which we solved by single anomalousdiffraction technique. It represents the largest structure (1,149 amino acid residues per molecule, 12 independent molecules per unit cell), which has been solved so far by single anomalousdiffraction technique. Tranhydroxylase is a heterodimer, with the active Mo-molybdopterin guanine dinucleotide (MGD)2 site in the ␣-subunit, and three [4FeO4S] centers in the ␤-subunit. The latter subunit carries a seven-stranded, mainly antiparallel ␤-barrel domain. We propose a scheme for the transhydroxylation reaction based on 3D structures of complexes of the enzyme with various polyphenols serving either as substrate or inhibitor.

Heterologous Expression and Identification of the Genes Involved in Anaerobic Degradation of 1,3-Dihydroxybenzene (Resorcinol) in Azoarcus anaerobius

“…All of the steps in the preparation of cell extracts were performed anaerobically. Cultures of A. anaerobius and T. aromatica transconjugants were harvested at an OD 578 of 0.3 and washed once under anoxic conditions with 100 ml of 50 mM potassium phosphate buffer (pH 7.0) as described previously (28). Unless used immediately, cell pellets were frozen in liquid N 2 and stored at Ϫ20°C.…”

“…Pyrogallol-phloroglucinol transhydroxylase is a soluble protein that catalyzes the conversion of pyrogallol to phloroglucinol (1,3,5-trihydroxybenzene). Its sequence is available (3), and its crystal structure was resolved as well (1,28). The holoenzyme contains a molybdenum ion coordinated to two molybdopterin guanidine dinucleotide cofactors in the large subunit and three four-iron, four-sulfur clusters in the small subunit (3,28).…”

Anaerobic Catabolism of Aromatic Compounds: a Genetic and Genomic View

“…5). The best match for the rhL and rhS gene products are the large and small subunits of the heterodimeric pyrogallolphloroglucinol transhydroxylase from Pelobacter acidigallici, a member of the dimethyl sulfoxide reductase family (244). As in the case of the transhydroxylase, the RhL and RhS subunits contain putative binding sites for two molybdopterin guanine dinucleotide cofactors and two iron-sulfur clusters, respectively (77).…”

“…1), which then becomes isomerized to phloroglucinol through a transhydroxylation reaction catalyzed by the cytoplasmic pyrogallol-phloroglucinol transhydroxylase (320). The amino acid sequence and three-dimensional structure of this transhydroxylase are known (11,244). The heterodimeric protein contains a molybdenum ion coordinated to two molybdopterin guanine dinucleotide cofactors in the large ␣-subunit and three [4Fe-4S] clusters in the small ␤-subunit.…”

“…The heterodimeric protein contains a molybdenum ion coordinated to two molybdopterin guanine dinucleotide cofactors in the large ␣-subunit and three [4Fe-4S] clusters in the small ␤-subunit. During the transhydroxylation reaction, the transferred hydroxyl group does not originate from water; instead, a cosubstrate such as 1,2,3,5-tetrahydroxybenzene is used without apparent electron transfer (244).…”