INTRODUCTION

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Pseudomonas pseudoalcaligenes JS45 (hereafter referred to as JS45) grows on nitrobenzene as the sole source of carbon, nitrogen, and energy (Fig. 1) (3, 10). 2-Aminophenol 1,6-dioxygenase catalyzes the cleavage of the aromatic ring of 2-aminophenol, yielding 2-aminomuconate 6-semialdehyde (2-AMS), which is unstable and spontaneously converts to picolinic acid (9, 10). Pseudomonas sp. strain AP-3 (16) degrades 2-aminophenol in an identical step to the ring fission of 2-aminophenol by JS45. The enzymes involved in metabolism after ring cleavage have been difficult to characterize biochemically, in part due to the instability of 2-AMS. Recently, we have demonstrated that in JS45, 2-aminophenol is degraded to pyruvate and acetaldehyde via a pathway similar to that of the meta cleavage of catechol (Fig. 1) (2, 3). In the pathway, 2-AMS is converted enzymatically to 2-aminomuconate in the presence of NAD⁺ by 2-AMS dehydrogenase (3).

View larger version (12K): [in this window] [in a new window]   FIG. 1.   Metabolic pathway for the degradation of nitrobenzene by P. pseudoalcaligenes JS45 (2, 3, 10).

2-AMS dehydrogenase was first investigated in the metabolism of tryptophan in mammalian tissues with 2-hydroxymuconic 6-semialdehyde (2-HMS) as a substrate (6, 11). Several genes of bacterial 2-HMS dehydrogenases have been sequenced (5, 8, 12, 18). However, only one 2-HMS dehydrogenase from P. putida has been purified and characterized, initially as a benzaldehyde dehydrogenase (7, 15). To investigate the properties of the bacterial 2-AMS dehydrogenase and to determine its relationship to 2-HMS as well as other semialdehyde dehydrogenases, purification, characterization, and sequence analysis of 2-AMS dehydrogenase from JS45 were performed in the present study.

	MATERIALS AND METHODS

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Bacterial strains and growth conditions. JS45 was grown with nitrobenzene as described previously (10). Escherichia coli DH5 was grown in Luria broth (Difco) with shaking or on Luria agar plates at 37°C. When necessary, ampicillin was added to a final concentration of 100 µg/ml.

Protein purification. All purification procedures were carried out at 4°C. Crude extracts were prepared as described previously (2). The crude extract (25 ml) was loaded onto a DEAE-Sepharose column (Pharmacia; 2.6 by 10 cm). The column was washed with 200 ml of 50 mM potassium phosphate buffer (pH 7.0), and proteins were eluted with a linear NaCl gradient (0 to 0.4 M in buffer, 400 ml at 2 ml/min). Fractions containing 2-AMS dehydrogenase activity were pooled, and proteins were fractionated by ammonium sulfate precipitation. The material that precipitated between 1.2 and 2.5 M ammonium sulfate was dissolved in 3.2 ml of buffer, loaded onto a Sephacryl S-300 gel filtration column (Pharmacia; 1.6 by 100 cm), and eluted with 50 mM potassium phosphate-0.1 M NaCl (pH 7.0, 1 ml/min). Active fractions were pooled and diluted with an equal volume of potassium phosphate buffer (pH 7.0) and loaded onto a Mono Q HR10/10 column (Pharmacia; 8 ml). The column was washed with 30 ml of 0.05 M NaCl in buffer. The active fractions were eluted around 0.21 M NaCl in a NaCl gradient (0.05 to 0.30 M NaCl in buffer, 90 ml at 1 ml/min). The enzyme was stored at 70°C and used for characterization studies.

Enzyme assays. 2-AMS dehydrogenase activity was routinely assayed by measurement of the decrease in absorbance of the substrate analog, 2-HMS, at 375 nm in the presence of 0.1 mM NAD⁺ in 100 mM potassium phosphate buffer (pH 7.5;  = 44,000 M¹) (11). The initial rate of dehydrogenation (in less than 1 min) was recorded. The physiological substrate, 2-AMS, was prepared in the reaction mixture by the addition of 2-aminophenol and excess partially purified 2-aminophenol 1,6-dioxygenase (3, 9). The activity on 2-AMS was determined by the increase in absorbance at 326 nm, concomitant with appearance of the product, 2-aminomuconate (2, 11). Activity on the other substrate analogs was measured by the increase in absorbance at 340 nm due to production of NADH. Protein concentrations were determined by the Coomassie Plus protein assay reagent from Pierce (Rockford, Ill.) by using bovine serum albumin as a standard.

Estimation of molecular mass. The subunit molecular mass of 2-AMS dehydrogenase was determined by comparison with protein molecular mass standards (low-range SigmaMarkers) on a sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis gel. The native molecular mass was determined by gel filtration on a Superdex 200 HR 10/10 column (Pharmacia) with 50 mM potassium phosphate-0.1 M NaCl (pH 7.0, 1 ml/min). The molecular size standards for gel filtration were ferritin (440 kDa), -amylase (200 kDa), alcohol dehydrogenase (150 kDa), and bovine serum albumin (66 kDa).

DNA and amino acid sequencing. The N-terminal amino acid sequence was determined at the Protein Core Facility of the University of Florida, Gainesville. The gene (designated amnC) encoding the enzyme was identified by matching the amino acid sequence of the purified enzyme with that of a deduced open reading frame located 258 bp downstream of the amnBA genes of 2-aminophenol 1,6-dioxygenase in E. coli clones (1a). The N-terminal part of the DNA sequence was determined at the Genetic Engineering Facility at the University of Illinois. The remainder of the sequence was determined with an ALF sequencer (Pharmacia) according to the manufacturer's instructions. Alignment of the sequence of the amnC product with those of other dehydrogenases was performed by using CLUSTALX (17) in the multiple-alignment mode with all parameters set to default. The GenBank accession number of the sequence is AF036343.

Chemicals. 2-HMS was enzymatically prepared from catechol by a method described previously (11) by using the resting cells of E. coli JM109/pDTG603 (20). All other chemicals were from Sigma (St. Louis, Mo.) or Aldrich (Milwaukee, Wis.), unless stated otherwise.

	RESULTS AND DISCUSSION

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Purification and molecular mass of 2-aminomuconic 6-semialdehyde dehydrogenase. A typical purification procedure (Table 1) yielded a 12.5-fold purification with a recovery of 37.5% of the 2-AMS dehydrogenase activity. Analysis of the purified protein by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis revealed a single band which corresponds to a molecular mass of 57 kDa. The amnC gene of 2-AMS dehydrogenase consists of 1,629 nucleotides, which would encode a protein of 542 amino acids with a molecular mass of 57.8 kDa. This subunit of 2-AMS is larger than that of 2-HMS dehydrogenase (51.5 to 51.7 kDa) (5, 8, 12, 18). Gel filtration chromatography of 2-AMS dehydrogenase revealed a native molecular mass of 160 kDa, suggesting that the enzyme is composed of three identical subunits. The active form of 2-HMS dehydrogenase from P. putida is a homodimer (15). Although there is no information on the native molecular mass and the subunit structure of 2-AMS dehydrogenase from cat liver, the quaternary structures of other eukaryotic aldehyde dehydrogenases are variable and the majority are homodimers or homotetramers (4). Because a homotrimeric structure is uncommon, more research is required to rigorously determine the native structure of the 2-AMS dehydrogenase.

Catalytic properties of the enzyme. The optimal pH for 2-AMS dehydrogenase activity was 7.3. Calculated from Lineweaver-Burk plots, at pH 7.5 and 25°C, the K_m for 2-HMS was 26 ± 2 µM (mean ± standard deviation) in the presence of 500 µM NAD⁺. The K_m for NAD⁺ was 74 ± 1 µM in the presence of 45 µM 2-HMS. The K_m values for cat liver 2-AMS dehydrogenase were 16 (2-HMS) and 19 (NAD⁺) µM (6). The 2-HMS dehydrogenase from P. putida had K_m values of 17 µM for 2-HMS and 330 µM for NAD⁺ (7). The high affinity of 2-AMS dehydrogenase for NAD⁺ might enhance the binding of NAD⁺ to the enzyme to facilitate the conversion of 2-AMS to 2-aminomuconate and to avoid unnecessary spontaneous conversion of 2-AMS to picolinate.

Substrate specificity. 2-AMS dehydrogenase was purified by using 2-HMS as a substrate analog. As shown in Fig. 2A, 2-aminophenol 1,6-dioxygenase cleaved 2-aminophenol (A₂₈₂) to 2-AMS (A₃₈₀), which spontaneously cyclized to picolinate (A₂₆₃). An absorbance maximum appeared at 326 nm rather than at 263 nm when 2-AMS dehydrogenase was included in the incubation mixture (Fig. 2B), indicating that 2-AMS was converted to 2-aminomuconate by 2-AMS dehydrogenase. The rapid spontaneous conversion of 2-AMS to picolinate made it impossible to measure the dehydrogenation reaction rate at a saturating (or stable) substrate concentration. By measuring the formation of 2-aminomuconate, the relative activity of 2-AMS dehydrogenase on 2-AMS was about 300% of the activity towards 2-HMS under the same conditions, indicating that 2-AMS is the physiological substrate for the enzyme.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Enzymatic formation of 2-aminomuconate from 2-aminophenol. (A) 2-Aminophenol (A282) was transformed to 2-AMS (A380) by 2-aminophenol 1,6-dioxygenase. 2-AMS was spontaneously converted to picolinic acid (A263). The reaction mixture contained 100 mM potassium phosphate (pH 7.5), 0.04 mM 2-aminophenol, 0.05 mg of partially purified dioxygenase per ml, 0.033 mM NAD+, and 0.133 mM pyruvate along with 4 U of lactate dehydrogenase/ml as an NAD+ regenerating system to eliminate the spectral interference of NADH with the absorbance of 2-aminomuconate during dehydrogenation. Recordings were taken at 0 time (curve 1), 10 s (curve 2), and later at 0.5-min intervals. (B) 2-Aminophenol was transformed to 2-aminomuconate (A326) in the presence of 2-aminophenol 1,6-dioxygenase and 2-AMS dehydrogenase. All the assay conditions were unchanged except for the presence of 2-AMS dehydrogenase (0.011 mg/ml).

Sequence analysis of 2-AMS dehydrogenase. AmnC has high amino acid sequence identity to 2-HMS dehydrogenases (43 to 55% to the first 329 amino acid residues) and somewhat less identity (33%) to the same region of 5-carboxymethyl-2-hydroxymuconic semialdehyde dehydrogenases (Fig. 3). The high sequence identity of the JS45 enzyme with 2-HMS dehydrogenases explains the ability of 2-AMS dehydrogenase to act on 2-HMS. It is clear from the alignment that 2-AMS dehydrogenase from JS45 is homologous with the 2-HMS dehydrogenases. Conserved residues are present at positions 255 (Glu) and 289 (Cys), and a putative NAD⁺ binding site at residues 231 to 238 is conserved (1, 5, 19). A second putative NAD⁺ binding site (GIGXXG) does not exist in 2-AMS dehydrogenase even though it is conserved in other 2-HMS dehydrogenases. This finding supports the argument of Nordlund and Shingler (12) that GXGXXG is not likely to be an NAD⁺ binding site.

View larger version (57K): [in this window] [in a new window]   FIG. 3.   CLUSTALX alignment of AmnC from JS45 (AmnC-JS45) with 2-HMS dehydrogenase XylG from Pseudomonas putida (XylG-pWW0) (5), XylG from Cycloclasticus oligotrophus (XylG-RB1) (18), DmpC from Pseudomonas strain CF600 (DmpC-CF600) (12), and PhnG from Pseudomonas sp. strain DJ77 (PhnG-DJ77) (8) and of 5-carboxymethyl-2-hydroxymuconic acid semialdehyde dehydrogenases HpcC from E. coli C (HpcC-EcoC) (14) and HpaE from E. coli W (HpaE-Eco11105) (13). Numeral symbols above positions 255 and 289 indicate conserved residues shown to be essential for catalysis (1, 4). FTGXTXXG (FTGXSXXG in AmnC) and GIGXXG above the aligned sequences indicate the locations of proposed NAD binding sites (4, 19). Asterisks below the aligned sequences indicate amino acids present in all seven sequences. Positions with colons below contain a residue of the strongly conserved groups in all seven sequences, and periods indicate more weakly conserved groups in all seven sequences.