Phosphorylation of phenol by phenylphosphate synthase: role of histidine phosphate in catalysis

Abstract

The anaerobic metabolism of phenol proceeds via carboxylation to 4-hydroxybenzoate by a two-step process involving seven proteins and two enzymes ("biological Kolbe-Schmitt carboxylation"). MgATP-dependent phosphorylation of phenol catalyzed by phenylphosphate synthase is followed by phenylphosphate carboxylation. Phenylphosphate synthase shows similarities to phosphoenolpyruvate (PEP) synthase and was studied for the bacterium Thauera aromatica. It consists of three proteins and transfers the beta-phosphoryl from ATP to phenol; the products are phenylphosphate, AMP, and phosphate. We showed that protein 1 becomes phosphorylated in the course of the reaction cycle by [beta-(32)P]ATP. This reaction requires protein 2 and is severalfold stimulated by protein 3. Stimulation of the reaction by 1 M sucrose is probably due to stabilization of the protein(s). Phosphorylated protein 1 transfers the phosphoryl group to phenolic substrates. The primary structure of protein 1 was analyzed by nanoelectrospray mass spectrometry after CNBr cleavage, trypsin digestion, and online high-pressure liquid chromatography at alkaline pH. His-569 was identified as the phosphorylated amino acid. We propose a catalytic ping-pong mechanism similar to that of PEP synthase. First, a diphosphoryl group is transferred to His-569 in protein 1, from which phosphate is cleaved to render the reaction unidirectional. Histidine phosphate subsequently serves as the actual phosphorylation agent.

FIG. 1.

Outlines of anaerobic metabolism of phenol, as studied for the denitrifying bacterium T. aromatica (20). The proposed reaction scheme for phenylphosphate synthase is hypothetical and is studied here. E₁, phenylphosphate synthase studied here; E₂, phenylphosphate carboxylase; Fd_red, reduced ferredoxin; E₃, 4-hydroxybenzoate-coenzyme A (CoA) ligase (AMP forming); E₄, 4-hydroxybenzoyl-CoA reductase (dehydroxylating); E₅, benzoyl-CoA reductase (aromatic ring reducing).

FIG. 2.

Partial sequence alignment of conserved histidine. Amino acids 550 to 578 of protein 1 from phenylphosphate synthase of T. aromatica are compared with protein 1 from phosphoenolpyruvate synthases of Corynebacterium glutamicum ATCC 13032 (BLAST gi 21323316), Clostridium acetobutylicum ATCC 824 (BLAST gi 15023398), and Bacillus subtilis subsp. subtilis strain 168 (BLAST gi 2634276). The amino acid positions of the shown fragments within the peptides are given by the numbers in front of the alignment. The conserved histidine residues are highlighted in white on a black background. The Basic Local Alignment Search Tool (BLAST) from NCBI was applied. For further information, see reference .

FIG. 3.

Requirements for phosphorylation of protein 1. In the phosphorylation assay, different combinations of proteins 1, 2, and 3 were incubated with 0.1 mM [β-³²P]ATP. After 30 min of incubation at 30°C, samples were retrieved and separated on SDS-PAGE gels, followed by phosphorimaging of labeled protein bands. Only the protein 1 band was labeled, and it is shown here in the autoradiogram of phosphorylated protein 1. Series a, absence of (−) sucrose; lane 1, 7 μg of protein 1; lane 2, 20 μg of protein 2; lane 3, 12.5 μg of protein 3; lane 4, 7 μg of protein 1 and 20 μg of protein 3; lane 5, 7 μg of protein 1 and 20 μg of protein 2; lane 6, 20 μg of protein 2 and 12.5 μg of protein 3; lane 7, 7 μg of protein 1 (1 nmol), 20 μg of protein 2 (5 nmol), and 12.5 μg of protein 3 (5 nmol). Series b, loaded proteins are as described for series a but with phosphorylation in the presence of (+) 1 M sucrose. For details of the phosphorylation assay, see Materials and Methods.

FIG. 4.

Dependence of protein 1 phosphorylation by [β-³²P]ATP on the concentration of ATP. Note that a constant amount of radioactivity (50,000 Bq per 50 μl assay mixture) was applied, i.e., the specific radioactivity of ATP decreased with increasing ATP concentration. The assay was carried out as described in the legend for Fig. 3 but using various ATP concentrations. Double determination, a deviation of ±5%.

FIG. 5.

Dephosphorylation of ³²P-labeled protein 1 by the substrate phenol and substrate analogues (each 1 mM). Lane 1, control in which phenol was not added; lanes 2 to 15, 1 mM of phenol and substrate analogues, respectively, were added as shown. The mixture (50 μl) containing 1 nmol of protein 1, 1 nmol of protein 2, 5 nmol of protein 3, and 1 mM [β-³²P]ATP (1,750 Bq/μl) was incubated at 30°C for 30 min. Then, substrate (analogue) was added and incubated further for 30 min at 30°C. For details, see Materials and Methods.

FIG. 6.

Time course of phosphorylation of protein 1 (20 μM) with 100 μM [β-³²P]ATP at 30°C and dephosphorylation by 1 mM phenol (A) at 0°C to slow down the reactions and (B) at 30°C. (C) The data were quantified by liquid scintillation counting of ³²P-labeled protein 1 spots which were excised from SDS-PAGE gels. The arrow indicates addition of phenol. ▴, dephosphorylation at 0°C; ▪, dephosphorylation at 30°C; •, phosphorylation at 30°C without the addition of phenol. The assay conditions were the same as those described in the legend for Fig. 5. Double determination, a deviation of ±5%.

FIG. 7.

Stability of phosphorylated protein. (A) Protein 1 was phosphorylated for 30 min at 30°C (pH 8.5) with [β-³²P]ATP in the presence of protein 2 and protein 3. Then, 0.1 volume of 1 M HCl, 1 M NaOH, and 1 M NH₂OH-KOH, pH 7.2, respectively, was added to the samples, followed by incubation at 40°C. At various time points, 5 μl of samples was withdrawn and analyzed for radioactive protein 1 by SDS-PAGE, followed by phosphorimaging. (B) Inhibition of phosphorylation of protein 1 by diethylpyrocarbonate (DEPC). Lane 1, control phosphorylation in 100 mM Tris-HCl, pH 8.5; lane 2, control phosphorylation in 100 mM HEPES-NaOH, pH 7.5; lane 3, phosphorylation mixture incubated with 100 mM diethylpyrocarbonate in HEPES-NaOH, pH 7.5, buffer for 10 min at 20°C, followed by addition of 5 μl of [β-³²P]ATP, with a final concentration of 0.1 mM (1,000 Bq/μl). After 30 min of incubation for phosphorylation at 30°C, 5 μl of samples was withdrawn, mixed with 10 μl of 2× SDS sample buffer, and analyzed by SDS-PAGE (10% polyacrylamide). For details, see Materials and Methods.

FIG. 8.

Amino acid sequence of protein 1 derived from the DNA sequence of open reading frame 1 in T. aromatica (GenBank accession no. AJ272115). Amino acid positions are given by the numbers at left. The peptides found by tandem MS after tryptic digestion of the expressed protein 1 are indicated by the line and tick marks just below the sequence. The dashed line indicates tryptic peptides found by HPLC only under alkaline conditions. The lower line indicates the peptides found after CNBr cleavage. Each peptide has been detected by its mass with high accuracy (0.6 ppm) and additionally identified by fragment ions. The phosphorylation site His-569 is shown in the bold frame.

FIG. 9.

HPLC chromatogram at pH 10 of the tryptic peptides derived from the protein 1 phosphorylation assay containing proteins 1, 2, and 3. (A) Base peak mass in the full scan range of m/z 250 to 1,800. (B) Chromatogram at m/z 951.9527 of the nonphosphorylated tryptic peptide with residues 558 to 575 of protein 1. (C) Chromatogram at m/z 991.9358 of the phosphorylated form of this peptide (found mass 1,981.8571, expected mass 1,981.8573). The column of 75-μm inside diameter had an emitter tip for nano-ESI and a length of 80 mm. Other details are described in Materials and Methods.

FIG. 10.

Proposed catalytic cycle of phenylphosphate synthase. The phosphorylation site is that of histidine 569. This hypothetical catalytic mechanism is analogous to the phosphoenolpyruvate synthase mechanism. The scheme shows the net phosphorylation of phenol catalyzed by combined action of protein 1 and protein 2 and the partial reaction, the exchange of free [¹⁴C]phenol, and the phenol moiety of phenylphosphate (exchange reaction), catalyzed by protein 1 alone. Note that the role of protein 3 is not explained by this scheme and is elusive.