Phenylphosphate Synthase: a New Phosphotransferase Catalyzing the First Step in Anaerobic Phenol Metabolism in Thauera aromatica

Abstract

The anaerobic metabolism of phenol in the beta-proteobacterium Thauera aromatica proceeds via paracarboxylation of phenol (biological Kolbe-Schmitt carboxylation). In the first step, phenol is converted to phenylphosphate which is then carboxylated to 4-hydroxybenzoate in the second step. Phenylphosphate formation is catalyzed by the novel enzyme phenylphosphate synthase, which was studied. Phenylphosphate synthase consists of three proteins whose genes are located adjacent to each other on the phenol operon and were overproduced in Escherichia coli. The promoter region and operon structure of the phenol gene cluster were Phenol is a natural substrate which is formed from a variety of natural compounds. Phenol arises from tyrosine by tyrosine phenol lyase, but phenol also arises during the degradation of many secondary phenolic plant constituents, notably in the course of the degradation of lignin and phenylpropanoid compounds. Besides phenol, there are many other phenolic compounds, both natural and synthetic ones. Their mineralization proceeds via completely different pathways, depending on whether oxygen is available or not. For instance, groundwater and landfills are free of oxygen. Therefore, anaerobic metabolism of phenolic compounds is of general interest, from both scientific and applied aspects.The initial steps in aerobic phenol metabolism are catalyzed by oxygenases. Phenol is oxidized to catechol (1,2-dihydroxybenzene) by phenol monooxygenases followed by oxygenolytic ring cleavage catalyzed by catechol dioxygenase. Hence, aerobic metabolism of phenol requires molecular oxygen for both ring hydroxylation and ring cleavage. In contrast to aerobic metabolism, anaerobic metabolism cannot rely on oxygen-and oxygenase-dependent steps. Therefore, anaerobic metabolism of phenol and related phenolic compounds promises unprecedented biochemistry. Anaerobic growth of pure cultures on phenol has been shown for sulfate-reducing (5), denitrifying (47,50,51), and iron-reducing (35) bacteria. The list of bacteria growing anaerobically with phenolic compounds is steadily growing (see references in reference 43). In all cases studied, anaerobic growth on phenol requires the presence of CO 2 (50); CO 2 is required as a cosubstrate for phenol carboxylation which results in the formation of 4-hydroxybenzoate. Phenol carboxylation has been known in chemistry for more than 100 years and is referred to as Kolbe-Schmitt carboxylation.Anaerobic phenol metabolism by pure cultures has been studied in some detail only in the denitrifying beta-proteobacterium Thauera aromatica (2, 3, 16, 24, 29-31, 43, 50, 51). It involves two initial steps (Fig.

Anaerobic Catabolism of Aromatic Compounds: a Genetic and Genomic View

“…This reaction requires protein 2, which resembles the N-terminal part of phosphoenolpyruvate synthase and is stimulated by an unknown mechanism by protein 3, which contains two cystathionine-␤-synthase domains but does not show significant similarity to known proteins. Phosphorylated protein 1 then transfers the phosphoryl group to the phenolic substrate (262,322). The genes encoding subunits 1 (orf1), 2 (orf2), and 3 (orf3) of phenylphosphate synthase are located adjacent to each other on a large operon that is phenol induced (Fig.…”

“…Phenol arises from tyrosine by tyrosine:phenol lyase (tyrosinase) but also arises during the degradation of many secondary phenolic plant constituents, notably in the course of the degradation of lignin and phenylpropanoid compounds. Phenol is also produced in large quantities by industrial activities (322).…”

“…catalyzed by a phenylphosphate synthase (protein E1) (322). Phenylphosphate synthase consists of three different subunits (proteins 1, 2, and 3) and transfers the ␤-phosphoryl group from ATP to phenol, generating phenylphosphate, AMP, and P i .…”

Anaerobic Metabolism of Catechol by the Denitrifying Bacterium Thauera aromatica —a Result of Promiscuous Enzymes and Regulators?

“…Extracts of phenol-grown cells (Fig. 4, lane f) and catechol-grown cells (lane e) contained large amounts of a 67-kDa polypeptide, which most likely represented protein 1 of the three-component enzyme phenylphosphate synthase (22,30). In addition 53-, 54-, 18-, and 10-kDa polypeptides, presumably representing the four subunits of phenylphosphate carboxylase, were induced (32).…”

“…The tubes were incubated at 30°C with 1 mM substrates, and after 0. 5,5,10,15,20,30, and 60 min, 1-ml samples were removed anaerobically and immediately cooled on ice. The cells were precipitated by centrifugation (4°C, 35,000 ϫ g, 10 min), and the concentrations of the substrates in the supernatants were determined by spectroscopic analysis (16,19) using overlay spectra.…”