Phenylphosphate carboxylase: a new C-C lyase involved in anaerobic phenol metabolism in Thauera aromatica

Abstract

The anaerobic metabolism of phenol in the beta-proteobacterium Thauera aromatica proceeds via carboxylation to 4-hydroxybenzoate and is initiated by the ATP-dependent conversion of phenol to phenylphosphate. The subsequent para carboxylation of phenylphosphate to 4-hydroxybenzoate is catalyzed by phenylphosphate carboxylase, which was purified and studied. This enzyme consists of four proteins with molecular masses of 54, 53, 18, and 10 kDa, whose genes are located adjacent to each other in the phenol gene cluster which codes for phenol-induced proteins. Three of the subunits (54, 53, and 10 kDa) were sufficient to catalyze the exchange of 14CO2 and the carboxyl group of 4-hydroxybenzoate but not phenylphosphate carboxylation. Phenylphosphate carboxylation was restored when the 18-kDa subunit was added. The following reaction model is proposed. The 14CO2 exchange reaction catalyzed by the three subunits of the core enzyme requires the fully reversible release of CO2 from 4-hydroxybenzoate with formation of a tightly enzyme-bound phenolate intermediate. Carboxylation of phenylphosphate requires in addition the 18-kDa subunit, which is thought to form the same enzyme-bound energized phenolate intermediate from phenylphosphate with virtually irreversible release of phosphate. The 54- and 53-kDa subunits show similarity to UbiD of Escherichia coli, which catalyzes the decarboxylation of a 4-hydroxybenzoate derivative in ubiquinone (ubi) biosynthesis. They also show similarity to components of various decarboxylases acting on aromatic carboxylic acids, such as 4-hydroxybenzoate or vanillate, whereas the 10-kDa subunit is unique. The 18-kDa subunit belongs to a hydratase/phosphatase protein family. Phenylphosphate carboxylase is a member of a new family of carboxylases/decarboxylases that act on phenolic compounds, use CO2 as a substrate, do not contain biotin or thiamine diphosphate, require K+ and a divalent metal cation (Mg2+or Mn2+) for activity, and are strongly inhibited by oxygen.

FIG. 1.

Anaerobic metabolism of phenol in T. aromatica. E₁, phenylphosphate synthase; E₂, phenylphosphate carboxylase; E₃, 4-hydroxybenzoate CoA ligase; E₄, 4-hydroxybenzoyl-CoA reductase; E₅, benzoyl-CoA reductase. Fd_red, reduced ferredoxin. Brackets indicate an enzyme-bound phenolate intermediate.

FIG. 2.

Completed and revised organization of the cluster of genes involved in anaerobic metabolism of phenol and possibly other phenolic compounds in T. aromatica. The arrows indicate the direction of transcription. Genes coding for phenol-induced proteins are indicated by stars. The corresponding calculated masses of the deduced proteins are indicated below the ORFs. For comparison, a similar gene cluster of G. metallireducens is shown (ORFs gi|23053807 to gi|23053791). Similar ORFs or groups of ORFs are indicated by black, different shades of gray, and stripes. The large numbers indicate the ORF numbers of T. aromatica.

FIG. 3.

Purification of phenylphosphate carboxylase from 5 g (wet weight) of T. aromatica cells. (A) Purification of the core enzyme composed of a 53-kDa subunit (ORF4, β subunit), a 54-kDa subunit (ORF6, α subunit), and a 10-kDa subunit (ORF12, γ subunit). Note that the molecular masses of the subunits are the molecular masses deduced from the genes which matched those determined experimentally here well. A Coomassie blue-stained SDS-13.5% PAGE gel was used. Lanes 1 and 8, molecular mass standards (14, 29, 34, 45, 67, and 97 kDa); lane 2, cell extract of T. aro-matica grown on 4-hydroxybenzoate for comparison (20 μg of protein); lane 3, cell extract of T. aromatica grown on phenol (20 μg of protein); lane 4, DEAE-Sepharose fraction (16 μg of protein); lane 5, Butyl TSK-Sepharose fraction (12 μg of protein); lane 6, ammonium sulfate precipitate fraction (12 μg of protein); lane 7, Superdex 200 gel filtration fraction (7.5 μg of protein). Arrows: lane 4, γ subunit; lane 7, α, β, and γ subunits. (B) Purification of the 18-kDa subunit (ORF5, δ subunit). A Coomassie blue-stained SDS-13.5% PAGE gel was used. Lane 1, cell extract of T. aromatica grown on phenol (20 μg of protein); lane 2, DEAE-Sepharose fraction (16 μg of protein); lane 3, Butyl TSK-Sepharose fraction (12 μg of protein); lane 4, hydroxyapatite fraction (12 μg of protein); lane 5, Superdex 200 gel filtration fraction (5 μg of protein); lane 6, molecular mass standards (14, 29, 34, 45, 67, and 97 kDa). Arrow, γ subunit.

FIG. 4.

Inactivation of phenylphosphate carboxylation activity by oxygen (A) and dithionite (B). The decreases in activity after different periods of incubation with various concentrations of oxygen and dithionite are shown.

FIG. 5.

Overexpression of orf4, orf5, orf6, and orf12 in E. coli. A Coomassie blue-stained SDS-13.5% PAGE gel was used. Only the soluble protein fractions (20 μg of protein) are shown. Overproduced proteins are indicated by arrows. Lanes 1 and 6, molecular mass standards (14, 29, 34, 45, 67, and 97 kDa); lane 2, E. coli TUNER (negative control); lane 3, ORF4 (53 kDa); lane 4, ORF5 (18 kDa); lane 5, ORF6 (54 kDa) and ORF12 (10 kDa).

FIG. 6.

Possible role of ORF4, ORF5, ORF6, and ORF12 in the phenylphosphate carboxylation reaction and the CO₂ exchange reaction with the carboxyl group of 4-hydroxybenzoate. For details see the text.

FIG. 7.

Comparison of the organizations of genes involved in carboxylation of phenol in T. aromatica and in decarboxylation of vanillic acid (vdc) and 4-hydroxybenzoate in various organisms. Similar genes involved in decarboxylation of vanillic acid in E. coli and other organisms are not shown. Sequencing of the C. hydroxybenzoicum genes is not complete. Black arrows, UbiD-like; light gray arrows, UbiX-like; dark gray arrows, small subunit of the decarboxylases; striped arrows, small subunit of phenylphosphate carboxylase.

FIG. 8.

Amino acid sequence similarity of proteins of the UbiD type (A) and the UbiX type (B) of aryl (de-)carboxylases. For details see the text and Fig. 7. The PAM scale indicates the percentage of point accepted mutation. Geobacter801, GI:23053801; Geobacter804, GI:23053804.