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Biodegradation of phenanthrene by Alcaligenes sp. strain PPH: partial purification and characterization of 1-hydroxy-2-naphthoic acid hydroxylase

Jaigeeth Deveryshetty, Prashant S. Phale
DOI: http://dx.doi.org/10.1111/j.1574-6968.2010.02079.x 93-101 First published online: 1 October 2010

Abstract

Alcaligenes sp. strain PPH degrades phenanthrene via 1-hydroxy-2-naphthoic acid (1-H2NA), 1,2-dihydroxynaphthalene (1,2-DHN), salicylic acid and catechol. Enzyme activity versus growth profile and heat stability studies suggested the presence of two distinct hydroxylases, namely 1-hydroxy-2-naphthoic acid hydroxylase and salicylate hydroxylase. 1-Hydroxy-2-naphthoic acid hydroxylase was partially purified (yield 48%, fold 81) and found to be a homodimer with a subunit molecular weight of ∼34 kDa. The enzyme was yellow in color, showed UV-visible absorption maxima at 274, 375 and 445 nm, and fluorescence emission maxima at 527 nm suggested it to be a flavoprotein. The apoenzyme prepared by the acid–ammonium sulfate (2 M) dialysis method was colorless, inactive and lost the characteristic flavin absorption spectra but regained ∼90% activity when reconstituted with FAD. Extraction of the prosthetic group and its analysis by HPLC suggests that the holoenzyme contained FAD. The enzyme was specific for 1-H2NA and failed to show activity with any other hydroxynaphthoic acid analogs or salicylic acid. The Km for 1-H2NA in the presence of either NADPH or NADH remained unaltered (72 and 75 μM, respectively), suggesting dual specificity for the coenzyme. The Km for FAD was determined to be 4.7 μM. The enzyme catalyzed the conversion of 1-H2NA to 1,2-DHN only under aerobic conditions. These results suggested that 1-hydroxy-2-naphthoic acid hydroxylase is a flavoprotein monooxygenase specific for 1-H2NA and different from salicylate-1-hydroxylase.

Keywords
  • phenanthrene degradation
  • flavoprotein hydroxylases
  • 1-hydroxy-2-naphthoic acid hydroxylase
  • kinetic characterization
  • substrate specificity

Introduction

In bacteria, phenanthrene is metabolized to a key intermediate, 1-hydroxy-2-naphthoic acid (1-H2NA), which is further channelized to the central carbon pathway either via a ‘naphthalene route’ (Rogoff & Wender, 1957; Evans et al., 1965; Prabhu & Phale, 2003) or via a ‘phthalate route’ (Iwabuchi & Harayama, 1998; Deveryshetty, 2009; Deveryshetty & Phale, 2009). In the ‘naphthalene route’, 1-H2NA is metabolized via 1,2-dihydroxynaphthalene (1,2-DHN) and salicylic acid to catechol by a series of enzymatic steps similar to naphthalene metabolic pathway. Biochemical and genetic studies suggest that enzymes responsible for the conversion of naphthalene to salicylic acid could also transform phenanthrene to 1-H2NA (Menn et al., 1993; Kiyohara et al., 1994; Takizawa et al., 1994). Phenanthrene-degrading Pseudomonas putida strain BS202P1, which also metabolizes naphthalene, is reported to have a broad substrate-specific salicylate-1-hydroxylase which is also responsible for the conversion of 1-H2NA to 1,2-DHN (Balashova et al., 2001). However, the enzyme showed a higher catalytic efficiency for salicylic acid as compared to 1-H2NA. N-terminal amino acid sequence showed significant homology with salicylate-1-hydroxylases from other gram-negative bacteria (Balashova et al., 2001).

Soil isolate Alcaligenes sp. strain PPH degrades phenanthrene as the sole carbon source. The specific activity versus growth profile indicated the presence of two hydroxylases, salicylate-1-hydroxylase and 1-hydroxy-2-naphthoic acid hydroxylase, in this strain (Deveryshetty, 2009). Salicylate-1-hydroxylase from various bacterial sources have been characterized and reported to have wide substrate specificity (Yamamoto et al., 1965; Katagiri et al., 1966; White-Stevens et al., 1972; Tu et al., 1981; You & Roe, 1981; You et al., 1990; Bosch et al., 1999; Balashova et al., 2001; Pinyakong et al., 2003; Zhao et al., 2005; Jouanneau et al., 2007). The hydroxylation of 1-H2NA to 1,2-DHN is similar to that of salicylic acid to catechol. However, the enzyme specific for 1-H2NA has not been reported so far. The aim of the present study is to purify 1-hydroxy-2-naphthoic acid hydroxylase from Alcaligenes sp. strain PPH and study its kinetic properties and substrate specificity. Here, we report partial purification and characterization of 1-hydroxy-2-naphthoic acid hydroxylase from the phenanthrene-degrading Alcaligenes sp. strain PPH.

Materials and methods

Chemicals

Phenanthrene, 1-H2NA, salicylic acid, gentisic acid, 3-hydroxy-2-naphthoic acid, 2-hydroxy-1-naphthoic acid, 1-naphthol, 2-naphthol, 1-naphthoic acid, 2-naphthoic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 1,2-DHN, catechol, 3,4-dihydroxybenzoic acid, EDTA, 1′,10′-phenanthroline, 2,2′-dipyridyl, DEAE–Sephacel and Sephacryl S-200-HR were purchased from Sigma-Aldrich. All other chemicals used were of analytical grade and purchased locally.

Bacterial culture and growth conditions

Alcaligenes sp. strain PPH was grown on 150 mL minimal salt medium supplemented with phenanthrene (0.1%, crystals) or glucose (0.25%) in baffled Erlenmeyer flasks (capacity 500 mL) at 30 °C on a rotary shaker at 200 r.p.m. (Deveryshetty et al., 2007).

Whole-cell oxygen (O2) uptake

Cells grown on phenanthrene (0.1%, crystals) or salicylate (0.1%) or glucose (0.25%) were used to monitor the whole-cell O2 uptake. Rates were measured in the presence of various probable metabolic intermediates at 30 °C using Oxygraph (Hansatech, UK) fitted with Clark's O2 electrode as described (Deveryshetty et al., 2007).

Preparation of cell-free extract and enzyme assays

Cells grown on phenanthrene (0.1%, crystals) or salicylate (0.1%) or glucose (0.25%) were harvested by centrifugation (10 000 g for 10 min), washed twice with potassium phosphate buffer (KPi, 50 mM, pH 7.5) and cell-free extract was prepared as described (Deveryshetty et al., 2007).

1-Hydroxy-2-naphthoic acid hydroxylase and salicylate-1-hydroxylase were monitored using Oxygraph by measuring the rate of O2 utilization. The reaction mixture (1 mL) contained substrate (100 μM, 1-H2NA or salicylate), NAD(P)H (300 μM), FAD (5 μM), an appropriate amount of enzyme (0.1 mg) and KPi buffer (50 mM, pH 7.5). 1,2-Dihydroxynaphthalene dioxygenase (Swetha & Phale, 2005), catechol-2,3-dioxygenase (Kojima et al., 1961), catechol-1,2-dioxygenase (Hayaishi & Hoshimoto, 1950), gentisate dioxygenase (Harpel & Lipscomb, 1990) and 3,4-dihydroxybenzoate dioxygenase (Stanier & Ingraham, 1954) were monitored as described. Enzyme activities were expressed as units (nmol or μmol of the product formed or substrate disappeared, NADH formed or O2 consumed) min−1 mL−1. Specific activities were expressed as units: mg−1 protein. Protein concentration was determined by the method of Bradford (1976) using bovine serum albumin (BSA) as the standard.

Identification of metabolites from the spent medium, whole-cell biotransformation and bulk enzyme reaction

Metabolites from the spent medium were extracted with an equal volume of ethyl acetate, dried and concentrated. Whole-cell biotransformation using salicylaldehyde as substrate was performed as described (Deveryshetty & Phale, 2009). The reaction products were resolved by thin layer chromatography (TLC) (0.5-mm-thick silica gel-coated glass plates) using the solvent system hexane : chloroform : acetic acid (7 : 3 : 1; v/v/v) and identified by comparing Rf and UV fluorescence properties at 254 nm with those of authentic compounds.

To identify the reaction product of 1-hydroxy-2-naphthoic acid hydroxylase, bulk enzyme reactions were performed under aerobic and anaerobic conditions using Thunberg's tube at 30 °C for 3 h. The reaction mixture (10 mL) contained KPi buffer (50 mM, pH 7.5), 1-H2NA (100 μM), NADPH (200 μM), FAD (5 μM) and an appropriate amount of enzyme. The reaction metabolites were isolated and identified as described earlier.

Purification of 1-hydroxy-2-naphthoic acid hydroxylase

1-Hydroxy-2-naphthoic acid hydroxylase was partially purified from Alcaligenes sp. strain PPH. All steps were carried out at 4 °C or on ice. Activities of both 1-hydroxy-2-naphthoic acid hydroxylase and salicylate-1-hydroxylase were monitored during all steps of purification.

Preparation of cell-free extract

Cells grown on phenanthrene (0.1%, culture vol. 10 L) were harvested, washed twice with Buffer A [KPi (20 mM, pH 7.5), glycerol (5%), 1-H2NA (0.1 mM), FAD (5 μM) and dithiothreitol (2 mM)] and resuspended in the ice-cold Buffer A (7.5 g in 30 mL). Cells were disrupted using an ultrasonic processor (GE130) on ice, with 10 cycles of 20 pulses each (1 s pulse, 1 s interval, cycle duration 40 s, output of 20 W, 3-min interval between two cycles). The supernatant obtained after centrifuging the cell homogenate at 50 000 g for 1 h was referred to as the cell-free extract.

Heat treatment

The cell-free extract was incubated at 60 °C in water bath in the presence of 1-H2NA (1 mM) with intermittent gentle shaking. After 5 min of incubation, the enzyme was immediately transferred on to ice. Denatured proteins were removed by centrifugation at 35 000 g for 30 min. The supernatant was dialyzed (a membrane cutoff of 12 kDa) against Buffer A and processed further.

Ammonium sulfate fractionation

The dialyzed heat-treated supernatant was brought to 0–30%, followed by 30–50% saturation by the addition of solid ammonium sulfate (over a period of ∼1 h), incubated for 30 min on ice with constant slow stirring and centrifuged at 35 000 g for 30 min at 4 °C. The pellet was suspended in a minimum volume of Buffer A and dialyzed against 500 mL of Buffer A for 3 h. The enzyme activity was present in 30–50% ammonium sulfate fraction.

DEAE–Sephacel chromatography

The dialyzed ammonium sulfate (30–50%) fraction was loaded onto a DEAE–Sephacel column (100 × 18 mm; bed vol. 19 mL) equilibrated with Buffer A. The column was washed extensively with Buffer B (Buffer A containing 0.15 M ammonium sulfate, 200 mL) and the enzyme was eluted with a linear gradient of ammonium sulfate (0.15–0.75 M in 100 mL) at a flow rate of 30 mL h−1. The enzyme was eluted as a single sharp peak between 0.22 and 0.4 M. Fractions containing activity>50 nmol O2 consumed min−1 mL−1 were pooled, dialyzed against Buffer A and used for further biochemical and kinetic characterization.

Determination of molecular weight

The subunit molecular weight of the enzyme was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12%) as described (Laemmli, 1970). The native molecular weight was determined using Sephacryl S-200-HR gel filtration chromatography. The column (600 × 12 mm; bed 60 mL; void 25 mL; flow rate of 3.5 mL h−1) was equilibrated with Buffer C [KPi (50 mM, pH 7.5) containing glycerol (5%) and dithiothreitol (2 mM)] and calibrated with standard molecular weight marker proteins (kDa): β-amylase (200), alcohol dehydrogenase (150), BSA (66) and carbonic anhydrase (29).

Spectroscopic characterization

To determine the spectroscopic properties, the partially purified enzyme (0.14 mg mL−1) was dialyzed against Buffer C for 5 h. The UV-visible absorption spectrum, in the presence and absence of sodium dithionite (1 mM), was recorded in the range of 200–700 nm (Lambda 35; Perkin-Elmer). The fluorescence emission spectrum of the enzyme (0.14 mg mL−1) was recorded by exciting it at 450 nm using a fluorescence spectrometer (Jasco V-750).

Apoenzyme preparation

The apoenzyme was prepared using the acid–ammonium sulfate method (Elmorsi & Hopper, 1977). The partially purified enzyme prepared (0.14 mg mL−1) was dialyzed against KPi buffer (50 mM, pH 5.5) containing (NH4)2SO4 (2 M), glycerol (5%) and dithiothreitol (2 mM) for 24 h at 4 °C. Both UV-visible and fluorescence spectral properties were monitored to confirm the apoenzyme preparation.

Identification of the prosthetic group

The prosthetic group was extracted by treating the holoenzyme (50 μg mL−1, 1 mL) with perchloric acid (10 μL of 70%) on ice for 5 min, followed by centrifugation at 22 000 g at 4 °C. The supernatant (40 μL) was subjected to HPLC (Jasco 1100 series) using an RP-C18 column (250 × 4 mm). A chromatogram was developed using an isocratic solvent system consisting of methanol (40%) and ortho-phosphoric acid (10 mM, 60%; v/v) in water. The eluent was identified by comparing the retention time and UV-visible spectral properties with that of the authentic FAD (retention time, 3.62 min) and FMN (retention time, 4.85 min) treated under the same conditions.

Kinetic constants

Kinetic constants were determined by measuring the initial reaction velocities with varying concentrations of 1-H2NA (10–800 μM), NAD(P)H (30–800 μM) or FAD (1–200 μM) using Oxygraph. The kinetic constants (Km and Vmax) were determined by plotting the enzyme activities versus substrate concentrations. All kinetic experiments were repeated twice with five different enzyme preparations. SDs observed between different sets of experiments are indicated appropriately. The kinetic data for 1-H2NA and NAD(P)H were fitted with (Vmax[S]n/Kmn+[S]n), while that for FAD were fitted with (Vmax[S]/Km+[S]).

Results

Metabolism of phenanthrene by Alcaligenes sp. strain PPH

Phenanthrene-grown culture showed a bright orange color in the early-log phase (9 h), which subsided and turned to pale green as it entered the stationary phase (30 h). Metabolites from the early-log (9 h), mid-log (18 h) and stationary (30 h) phase culture were extracted, resolved by TLC and identified by comparing their Rf values and fluorescence properties with those of authentic compounds. Three metabolite spots were detected in the spent medium of the early-log phase culture, which were identified as 1-H2NA, 1,2-DHN and salicylic acid (Rf values 0.95, 0.11 and 0.9, respectively). The spent medium of the late-log phase culture showed two spots corresponding to 1-H2NA and salicylic acid; while a single spot, salicylic acid, was identified in the stationary phase culture. Phenanthrene-grown cells were able to transform salicylaldehyde to salicylic acid and catechol (Rf values 0.9 and 0.37, respectively). Phenanthrene- and salicylic acid-grown cells showed comparable and significant O2 uptake on phenanthrene, 1-H2NA, 1,2-DHN, salicylaldehyde, salicylic acid and catechol (Table 1). Cells failed to respire on o-phthalic acid and 3,4-dihydroxybenzoic acid (Table 1). The cell-free extract prepared from phenanthrene-grown cells showed activities of 1-hydroxy-2-naphthoic acid hydroxylase, 1,2-dihydroxynaphthalene dioxygenase, salicylate-1-hydroxylase and catechol-2,3-dioxygenase (Table 2), while salicylic acid-grown cells showed comparatively reduced activities for all enzymes and significantly lower activity of 1-hydroxy-2-naphthoic acid hydroxylase (Table 2). The cell-free extract prepared from naphthalene-grown cells of P. putida strain CSV86 (this strain does not degrade phenanthrene or 1-H2NA, Mahajan et al., 1994) showed sevenfold less activity of salicylate-1-hydroxylase with 1-H2NA (53 nmol min−1 mg−1) as compared with salicylic acid (362 nmol min−1 mg−1) as substrate. The enzyme preparation from strain PPH failed to show activity of gentisic- and 3,4-dihydroxybenzoic acid dioxygenase (Table 2). Time-dependent spectral changes of catechol dioxygenase reaction showed an increase in A375 nm (Deveryshetty, 2009), indicating the formation of 2-hydroxymuconic semialdehyde due to meta-ring cleavage of catechol by catechol-2,3-dioxygenase (Kojima et al., 1961; Nozaki et al., 1963). Specific activity versus growth profiles showed maximum activity of 1-hydroxy-2-naphthoic acid hydroxylase and 1,2-dihydroxynaphthalene dioxygenase at 18 h, and maximum activity of catechol-2,3-dioxygenase at 21 h (Deveryshetty, 2009). Salicylate-1-hydroxylase activity was detectable, but at low levels. Cells grown on glucose showed neither O2 uptake nor enzyme activities in the cell-free extract (Deveryshetty, 2009), indicating that the enzymes of the pathway are inducible.

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Table 1

Whole-cell O2 uptake on various probable metabolites of phenanthrene degradation by Alcaligenes sp. strain PPH

SubstratesOxygen uptake (nmol min−1 mg−1 wet cells)
PhenanthreneSalicylic acid
Phenanthrene11.97.5
1-H2NA3.93.0
1,2-DHN5.14.2
Salicylaldehyde7.24.4
Salicylic acid0.91.7
Catechol2517.6
o-Phthalic acidNDND
3,4-Dihydroxybenzoic acidNDND
Gentisic acid0.20.6
  • * Cells were grown on the respective carbon source till late-log phase.

  • ND, not detected by this method.

  • Values are corrected for endogenous respiration. Each experiment was performed at least five times and enzyme activity was measured in triplicate. The average values are depicted in the table. The observed SD was ± 10%.

View this table:
Table 2

Activity of various enzymes in cell-free extract of Alcaligenes sp. strain PPH

EnzymesSpecific activity (nmol min−1 mg−1 protein)
PhenanthreneSalicylic acid
1-Hydroxy-2-naphthoic acid hydroxylase87.73.1
1,2-Dihydroxynaphthalene dioxygenase129.749.6
Salicylate-1-hydroxylase12.71.7
Catechol-2,3-dioxygenase17341133
Catechol-1,2-dioxygenaseNDND
3,4-Dihydroxybenzoic acid dioxygenaseNDND
Gentisate dioxygenaseNDND
  • * Cells were grown on the respective carbon source till late-log phase.

  • ND, not detected by this method.

  • Activities were monitored by polarographic method as described in Materials and methods. Each experiment was performed at least five times and enzyme activity was measured in triplicate. The average values are depicted in the table. The observed SD was ± 5%.

Partial purification of 1-hydroxy-2-naphthoic acid hydroxylase

1-Hydroxy-2-naphthoic acid hydroxylase in the cell-free extract was stabilized by 1-H2NA (0.1 mM), FAD (5 μM), dithiothreitol (2 mM) and glycerol (5%). Interestingly, the enzyme showed stability at 60 °C for 5 min in the presence of 1-H2NA, while the activity of salicylate-1-hydroxylase was lost, suggesting the presence of two distinct enzymes in the strain PPH. Using heat treatment, ammonium sulfate fractionation and DEAE anion-exchange chromatography, 1-hydroxy-2-naphthoic acid hydroxylase was partially purified (81-fold, with a 48% yield and a specific activity of 1518 nmol min−1 mg−1 protein) from phenanthrene-grown cells of Alcaligenes sp. strain PPH (Table 3). Native-PAGE analysis showed a prominent band of lower mobility and two minor contaminating bands with higher mobility (Fig. 1a). SDS-PAGE analysis showed a progressive enrichment of a protein band of ∼34 kDa (Fig. 1b). Additional purification steps such as hydrophobic (Phenyl- and Octyl-Sepharose) or gel filtration chromatography led to the total or a significant (∼70%) loss of activity, respectively, without achieving any further purification. The native molecular weight was determined to be 66 kDa, suggesting that the enzyme is probably a homodimer. The partially purified enzyme retained 100% activity when stored at −20 °C for 60 days in the presence of stabilizers such as 1-H2NA (0.1 mM), FAD (5 μM), dithiothreitol (2 mM) and glycerol (5%). Repeated freezing and thawing led to inactivation of the enzyme.

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Table 3

Partial purification of 1-hydroxy-2-naphthoic acid hydroxylase from Alcaligenes sp. strain PPH

StepTotal volume (mL)Total protein (mg)Total activitySpecific activityFoldYield
Cell-free extract41450844618.81100
Heat treatment40341782022.91.2292.6
Ammonium sulfate5.268.2239934.51.8328.4
DEAE–Sephacel402.66404015188148
  • * nmol O2 consumed min−1 mL−1 activity measured using NADPH as electron donor.

  • nmol O2 consumed min−1 mg−1 protein.

Figure 1

Purification of 1-hydroxy-2-naphthoic acid hydroxylase: (a) Native-PAGE (10%) analysis of a partially purified 1-hydroxy-2-naphthoic acid hydroxylase (DEAE-eluted pool). (b) SDS-PAGE analysis of 1-hydroxy-2-naphthoic acid hydroxylase purity at different stages of purification. Lanes (each lane contains 5 μg protein): 1, cell-free extract; 2, heat treatment; 3, ammonium sulfate fraction; 4, DEAE-eluted pool; M, molecular weight markers [markers (kDa): phosphorylase B (97.4), BSA (66), ovalbumin (43), carbonic anhydrase (29) and soybean trypsin inhibitor (20)]. The band corresponding to the enzyme is marked with an arrow head in (a) and (b).

Characterization of 1-hydroxy-2-naphthoic acid hydroxylase

Spectral properties

The partially purified enzyme was yellow in color and UV-visible spectrum yielded absorption maxima at 274, 375 and 445 nm (Fig. 2a). Addition of sodium dithionite (1 mM) resulted in the disappearance of the absorbance peak at 445 nm (Fig. 2a, inset). Further excitation of the enzyme at 450 nm yielded an emission maximum at 527 nm (Fig. 2b), suggesting that the enzyme probably has the flavin moiety.

Figure 2

Spectral properties of 1-hydroxy-2-naphthoic acid hydroxylase: (a) UV-visible spectrum of the holoenzyme and the apoenzyme (0.14 mg mL−1). Inset: magnified absorption spectra of the oxidized enzyme (solid line), which disappeared when treated with dithionite (dotted line), and the apoenzyme (dashed line). (b) Fluorescence emission spectrum for 1-hydroxy-2-naphthoic acid hydroxylase excited with 450-nm wavelength.

Coenzyme requirement and prosthetic group identification

The enzyme showed optimum activity at pH 7.5. The effect of various coenzymes and prosthetic groups on the enzyme activity is summarized in Table 4. In the absence of 1-H2NA, the enzyme failed to consume O2, suggesting the absence of nonspecific NAD(P)H oxidase activity (Table 4). The enzyme showed maximum activity in the presence of FAD and NADPH over any other combination tested (Table 4). The apoenzyme (FAD-free protein) prepared by the acid–ammonium sulfate dialysis method was colorless and inactive, and UV-visible absorption spectrum showed no absorption peaks at 375 and 445 nm (Fig. 2a). The activity of the apoenzyme could be restored to 92% by addition of FAD in the presence of NADPH as compared with FMN (Table 4). HPLC analysis of the flavin moiety extracted from the holoenzyme showed a retention time of 3.68 min, which corresponded with that of authentic FAD (3.62 min). Various metal ions (1 mM) such as Fe+2, Fe+3, Mg+2, Mn+2, Ca+2, Zn+2 and Cu+2 and metal chelators (1 mM) such as EDTA, α,α-dipyridyl and 1′,10′-phenanthroline failed to enhance or inhibit the activity of the enzyme.

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Table 4

Coenzyme requirement of 1-hydroxy-2-naphthoic acid hydroxylase

Reaction constituentsSpecific activity (nmol min−1 mg−1 protein)Activity (%)
(a) Holoenzyme
1-H2NAND
NAD(P)H+FAD/FMNND
1-H2NA+NADPH390072
1-H2NA+NADH236743.8
1-H2NA+NADPH+FAD5400100
1-H2NA+NADH+FAD373369
1-H2NA+NADPH+FMN486790
1-H2NA+NADH+FMN356766
(b) Reconstitution of apoenzyme activity
Holoenzyme+NADPH+FAD3866100
Apoenzyme+NADPHNDND
Apoenzyme+NADPH+FAD354392
Apoenzyme+NADPH+FMN198351
  • * Activity of holoenzyme before dialyzing.

  • Apoenzyme was prepared by dialyzing holoenzyme as described in Materials and methods.

  • ND, not detected.

Substrate specificity and reaction product identification

The activity of 1-hydroxy-2-naphthoic acid hydroxylase on various mono- and diaromatic compounds was monitored. Enzyme showed activity on 1-H2NA, but failed to show activity with 3-hydroxy-2-naphthoic acid, 2-hydroxy-1-naphthoic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 1-naphthol, 2-naphthol, 1-naphthoic acid, 2-naphthoic acid, salicylic acid, gentisic acid or catechol as substrate. These results suggest that the enzyme is highly specific for 1-H2NA.

TLC analysis of the enzyme reaction product obtained under aerobic conditions yielded two spots (Rf=0.95, blue fluorescence with quench in center and Rf=0.11, greenish black quench), which were identified as 1-H2NA and 1,2-DHN, respectively, by comparing with authentic compounds. Under anaerobic conditions, a single spot (Rf=0.95) corresponding to substrate 1-H2NA was observed. These results suggest that the enzyme catalyzes the conversion of 1-H2NA to 1,2-DHN in the presence of molecular O2, indicating the oxygenase nature of the enzyme.

Kinetic constants

Saturation plots for 1-hydroxy-2-naphthoic acid hydroxylase with 1-H2NA, NADPH and NADH were sigmoidal (Deveryshetty, 2009), suggesting that the enzyme probably exhibits cooperativity (Table 5). The Km for the substrate 1-H2NA remained unaltered in the presence of NADPH or NADH (Table 5). The enzyme showed similar Km for NADPH and NADH (Table 5). The saturation plot for FAD was hyperbolic and Km was determined to be 4.7 μM (Table 5).

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Table 5

Kinetic constants for partially purified 1-hydroxy-2-naphthoic acid hydroxylase

ConditionsV max (μmol min−1 mg−1 protein)K m (μM)Hill coefficient (n)
1-H2NA with NADPH1.10 ± 0.0871.6 ± 4.96
1-H2NA with NADH0.90 ± 0.0875.5 ± 4.98
NADPH0.96 ± 0.0384.6 ± 6.68
NADH0.88 ± 0.1487 ± 11.58
FAD2.35 ± 1.804.7 ± 0.6

Discussion

Alcaligenes sp. strain PPH degrades phenanthrene, hydroxybenzoates (o-, m- and p-) and o-phthalate (Deveryshetty et al., 2007). Based on metabolic analysis, the proposed pathway for phenanthrene degradation is: phenanthrene →→ 1-H2NA → 1,2-DHN →→ salicylaldehyde → salicylic acid → catechol. The steps involved in the metabolism of 1-H2NA to salicylic acid are similar to that involved in naphthalene degradation and hence referred to as the ‘naphthalene route’. The generated catechol enters the central carbon cycle via the meta ring-cleavage pathway. Organisms capable of degrading phenanthrene via the ‘naphthalene route’ have the ability to degrade naphthalene (Davies & Evans, 1964; Evans et al., 1965; Menn et al., 1993; Sanseverino et al., 1993; Kiyohara et al., 1994; Takizawa et al., 1994; Yang et al., 1994). Interestingly, strain PPH failed to metabolize naphthalene as the carbon source; this could be due to lack of naphthalene dioxygenase or the presence of highly specific phenanthrene dioxygenase in this strain. Compared to salicylate, phenanthrene-grown cells showed higher specific activity of 1-hydroxy-2-naphthoic acid hydroxylase (Table 2). As observed for several aromatic degradative pathways (Grund et al., 1990; Gescher et al., 2002; Phale et al., 2007; Swetha et al., 2007; Deveryshetty & Phale, 2009), enzymes of phenanthrene degradation in strain PPH were also found to be inducible in nature. The upper-pathway enzymes of naphthalene degradation (naphthalene to salicylic acid) have been proposed to be involved in the conversion of phenanthrene to 1-H2NA and anthracene to 2-hydroxy-1-naphthoic acid (Menn et al., 1993). Further, 1-H2NA was metabolized to 1,2-DHN by salicylate-1-hydroxylase and reported to have broad substrate specificity (Balashova et al., 2001). In Alcaligenes sp. strain PPH, enzyme induction pattern and heat stability studies suggested the existence of two different enzymes, 1-hydroxy-2-naphthoic acid hydroxylase and salicylate-1-hydroxylase, responsible for the conversion of 1-H2NA to 1,2-DHN and salicylic acid to catechol, respectively. The enzyme responsible for the hydroxylation of 1-H2NA has not been reported so far. This is the first study reporting the existence of 1-hydroxy-2-naphthoic acid hydroxylase.

The property of heat stability helped to resolve 1-hydroxy-2-naphthoic acid hydroxylase from salicylate hydroxylase and was exploited to partially purify the protein (Table 3). The enzyme was yellow in color and showed characteristic flavoprotein absorption spectrum (Fig. 2), as observed for several other hydroxylases (Yamamoto et al., 1965; Hesp & Calvin, 1969; White-Stevens & Kamin, 1972). The flavin moiety appears to be loosely associated with the enzyme as it can be removed completely by acid–ammonium sulfate dialysis as well as partially by gel filtration chromatography. The ability to restore the activity of the apoenzyme and identification by HPLC analysis from the holoenzyme suggests that the enzyme contains FAD as the prosthetic group (Table 4). Loosely bound FAD as the prosthetic group has been reported for several flavin hydroxylases (Takemori et al., 1969; Strickland & Massey, 1973; Elmorsi & Hopper, 1977; Wang et al., 1984; Tanner & Hopper, 2000). The enzyme could accept both NADPH and NADH as an external electron donor and does not show nonspecific NAD(P)H oxidase activity. External addition of metal ions and chelators has no effect on the activity. The homodimeric nature of the enzyme (subunit molecular weight of 34 kDa) suggests that 1-hydroxy-2-naphthoic acid hydroxylase is a FAD-containing single-component hydroxylase. The molecular mass of the single component system salicylate-1-hydroxylases are reported to be in the range of 38–57 kDa and are either monomers or dimers (Yamamoto et al., 1965; White-Stevens & Kamin, 1972; You et al., 1990; Balashova et al., 2001). A three-component salicylate-1-hydroxylase consisting of an oxygenase, a ferredoxin and a reductase has also been reported (Pinyakong et al., 2003; Jouanneau et al., 2007).

Flavin hydroxylases have been reported to accept electrons from NADH, NADPH or both (Ohta & Ribbons, 1976; Beadle & Smith, 1982; Van Berkel & Van Den Tweel, 1991; Swetha et al., 2007). Similarly, 1-hydroxy-2-naphthoic acid hydroxylase accepted electrons from both NADPH and NADH. The kinetic constants for NADPH or NADH clearly indicate that both electron donors are equally preferred by the enzyme (Table 5). The affinity for 1-H2NA (Km) remained unchanged, irrespective of the electron donor used. The enzyme saturation profiles with 1-H2NA, NADPH or NADH were sigmoidal, suggesting a regulatory role of this enzyme in the phenanthrene degradation pathway. A similar kinetic property has been reported for 3-hydroxybenzoate 6-hydroxylase from Klebsiella pneumoniae (Suarez et al., 1995), but not for salicylate hydroxylases so far. 1-Hydroxy-2-naphthoic acid hydroxylase from strain PPH failed to show the conversion of 1-H2NA to 1,2-DHN under anaerobic conditions, suggesting that the enzyme belongs to the oxygenase group. A majority of flavin hydroxylases, including salicylate hydroxylases, have been reported to be exhibiting broad substrate specificity (Beadle & Smith, 1982; Locher et al., 1991; Xun et al., 1992; Suske et al., 1997; Eppink et al., 2000). 1-Hydroxy-2-naphthoic acid hydroxylase from strain PPH was specific to 1-H2NA and failed to show activity on 1-H2NA analogs and salicylate. Flavoprotein hydroxylases with limited substrate have also been reported (Hosokawa & Stanier, 1966; Van Berkel & Van Den Tweel, 1991; Suarez et al., 1995; Haigler et al., 1996; Swetha et al., 2007). Unlike previously reported broad substrate-specific salicylate-1-hydroxylase from P. putida strain BS202P1 and P. putida strain S1 (Balashova et al., 2001), the present study suggests that strain PPH has two distinct and specific hydroxylases: 1-hydroxy-2-naphthoic acid hydroxylase and salicylate-1-hydroxylase.

In conclusion, the observed properties suggest that 1-hydroxy-2-naphthoic acid hydroxylase from Alcaligenes sp. strain PPH is a heat-stable, single-component flavoprotein aromatic hydroxylase specific for 1-H2NA.

Acknowledgements

J.D. thanks CSIR, Government of India, for a Senior research fellowship and P.P. thanks BRNS, DAE, Government of India, for the research grant.

Footnotes

  • Editor: Dieter Jahn

References

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