Functional Characterization of a Novel Member of the Amidohydrolase 2 Protein Family, 2-Hydroxy-1-Naphthoic Acid Nonoxidative Decarboxylase from Burkholderia sp. Strain BC1

ABSTRACT

The gene encoding a nonoxidative decarboxylase capable of catalyzing the transformation of 2-hydroxy-1-naphthoic acid (2H1NA) to 2-naphthol was identified, recombinantly expressed, and purified to homogeneity. The putative gene sequence of the decarboxylase (hndA) encodes a 316-amino-acid protein (HndA) with a predicted molecular mass of 34 kDa. HndA exhibited high identity with uncharacterized amidohydrolase 2 proteins of various Burkholderia species, whereas it showed a modest 27% identity with γ-resorcylate decarboxylase, a well-characterized nonoxidative decarboxylase belonging to the amidohydrolase superfamily. Biochemically characterized HndA demonstrated strict substrate specificity toward 2H1NA, whereas inhibition studies with HndA indicated the presence of zinc as the transition metal center, as confirmed by atomic absorption spectroscopy. A three-dimensional structural model of HndA, followed by docking analysis, identified the conserved metal-coordinating and substrate-binding residues, while their importance in catalysis was validated by site-directed mutagenesis.

IMPORTANCE Microbial nonoxidative decarboxylases play a crucial role in the metabolism of a large array of carboxy aromatic chemicals released into the environment from a variety of natural and anthropogenic sources. Among these, hydroxynaphthoic acids are usually encountered as pathway intermediates in the bacterial degradation of polycyclic aromatic hydrocarbons. The present study reveals biochemical and molecular characterization of a 2-hydroxy-1-naphthoic acid nonoxidative decarboxylase involved in an alternative metabolic pathway which can be classified as a member of the small repertoire of nonoxidative decarboxylases belonging to the amidohydrolase 2 family of proteins. The strict substrate specificity and sequence uniqueness make it a novel member of the metallo-dependent hydrolase superfamily.

INTRODUCTION

Decarboxylase is one of the most important classes of enzymes involved in a large variety of catabolic and anabolic pathways. The majority of the decarboxylases utilize an organic cofactor or a transition metal coupled with dioxygen to activate their substrates leading to the removal of carbon dioxide (1). However, there is a small group of transition metal-dependent decarboxylases that carry out decarboxylation of various aromatic acids in a nonoxidative manner. These nonoxidative decarboxylases act on various lignin-derived compounds, such as 4-hydroxybenzoic acid (2), (carboxy)vanillic acid (3, 4), protocatechuic acid (5), ferulic acid (6), p-coumaric acid (7), and estrogenic phthalate (8). Likewise, oxygen-independent decarboxylases are also involved in the 2-nitrobenzoic acid degradation pathway (9, 10), the tryptophan catabolic pathway (11), and the thymidine salvage pathway (12).

Nonoxidative decarboxylases, in general, can broadly be classified into two major groups depending on their oxygen sensitivity. Oxygen-sensitive decarboxylases, viz., 4-hydroxybenzoate decarboxylase (2), 3,4-dihydroxybenzoate decarboxylase (5), and indole-3-carboxylate decarboxylase (13), catalyze reversible reactions, including both carboxylation and decarboxylation. On the other hand, oxygen-insensitive decarboxylases, such as 2,3-dihydroxybenzoate decarboxylase (14), 5-carboxyvanillate decarboxylase (4), and 4,5-dihydroxyphthalate decarboxylase (8), have been reported to catalyze the decarboxylation reaction only. However, there are a few nonoxidative oxygen-insensitive decarboxylases, viz., γ-resorcylate decarboxylase (15), vanillate/4-hydroxybenzoate decarboxylase (16), and salicylate decarboxylase (17), that have been documented to catalyze reversible reactions.

Hydroxynaphthoates, such as 1-hydroxy-2-naphthoic acid (1H2NA), 2-hydroxy-1-naphthoic acid (2H1NA), and 3-hydroxy-2-naphthoic acid (3H2NA), are normally encountered during the bacterial degradation of polycyclic aromatic hydrocarbons, viz., phenanthrene, anthracene, and pyrene, and are metabolized either through ring cleavage (18–22) or by oxidative decarboxylation (23). In addition, decarboxylation of 1H2NA to 1-naphthol has been proposed, based on the identification of the later compound during degradation of phenanthrene in a few bacteria (24–26). Similarly, decarboxylation of phenanthrene-4,5-dicarboxylic acid to phenanthere-4-carboxylic acid has also been reported in the pathway of pyrene degradation (19). However, there is no documented report of any nonoxidatively decarboxylated product produced during the metabolism of 2H1NA or 3H2NA. Despite published reports of purification and characterization of several nonoxidative hydroxybenzoate decarboxylases (5, 14–16), there are no examples of any enzyme catalyzing nonoxidative decarboxylation of any of the hydroxynaphthoic acid isomers.

Previously, we reported a nonconventional degradation pathway of 2H1NA in Burkholderia sp. strain BC1 describing 2-naphthol, gentisaldehyde, and gentisic acid as pathway intermediates (27). Moreover, the presence of a strictly inducible nonoxidative decarboxylase was also observed in the cell extract of 2H1NA-grown culture catalyzing the enzymatic transformation of 2H1NA to 2-naphthol. In the present study, we describe a proteomic approach-based gene cloning and functional characterization of nonoxidative 2H1NA decarboxylase from Burkholderia sp. strain BC1. In addition, the roles of specific amino acid residues responsible for substrate binding and enzyme catalysis have been elucidated.

MATERIALS AND METHODS

Bacterial strains, plasmids, and culture conditions.

The strains and plasmids used in this study are listed in Table S1 in the supplemental material. Recombinant constructs in Escherichia coli [XL1-Blue and BL21(DE3)] were routinely grown and maintained in Luria-Bertani (LB) broth (per liter) containing 10 g of Bacto tryptone, 5 g of yeast extract, and 10 g of NaCl (pH 7.2) or on LB solid medium (1.8% [wt/vol] agar) at 37°C. Where appropriate, ampicillin (100 μg/ml), kanamycin (50 μg/ml), chloramphenicol (12.5 μg/ml), IPTG (isopropyl-β-d-thiogalactopyranoside; 0.1 to 1 mM), or X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; 20 μg/ml) was added. For expression cloning, pET28a (Novagen, Madison, WI) served as the expression vector.

Partial purification and gene identification of 2H1NA decarboxylase.

Native 2H1NA decarboxylase was purified from crude cell extract of BC1 cells grown for 16 h at 28°C in 4 liters of mineral salt medium (MSM) (20) containing 0.5 g/liter 2H1NA. Crude cell extract was prepared as described previously (27) and was then fractionated by sequential protein precipitation using ammonium sulfate. The 30 to 50% ammonium sulfate saturated fraction was centrifuged at 12,000 × g for 30 min, and the resulting pellet was dissolved in buffer A (50 mM K₂HPO₄-KH₂PO₄ buffer [pH 7.0]) and dialyzed against buffer B [50 mM K₂HPO₄-KH₂PO₄ buffer (pH 7.0) containing 0.8 M (NH₄)₂SO₄]. The dialyzed fraction was then loaded onto a column (2.5 by 10 cm), packed with phenyl-Sepharose 6 Fast Flow, preequilibrated with buffer B. The column was washed with 5 column volumes of buffer B, and then the adsorbed proteins were eluted in steps using 10 column volumes each of buffer A containing different concentrations of (NH₄)₂SO₄ (0.8 to 0.05 M). Finally, the column was washed with two column volumes of buffer A. All purification steps were carried out at 4°C or on ice under aerobic conditions. Fractions exhibiting 2H1NA decarboxylase activity were combined and dialyzed against buffer C (50 mM K₂HPO₄-KH₂PO₄ buffer [pH 7.0], 10% glycerol), concentrated by ultrafiltration (Millipore, Massachusetts), and stored at −80°C until further use. The purity of the protein fractions obtained after ammonium sulfate precipitation and hydrophobic interaction chromatography was evaluated by 12.5% SDS-PAGE analysis, followed by Coomassie blue staining in the presence of prestained protein molecular mass markers (Puregene; Genetix, India) by standard techniques. Protein quantification was performed according to the method of Bradford (28).

For the identification of the decarboxylase, tryptic digestion of the protein and subsequent extraction of peptides from SDS-PAGE gel matrices were carried out according to the methods described by Shevchenko et al. (29), followed by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) and tandem MS (MS/MS) analyses using an AutoFlex II (Bruker Daltonics, Germany) MALDI-tandem TOF (TOF/TOF) mass spectrometer equipped with a pulsed N₂ laser (λ = 337 nm, 50 Hz). The mass spectra were analyzed with Flex Analysis Software (version 2.4; Bruker; Daltonics). From the MS/MS data, partial amino acid sequences of the peptides were determined using PEAKS studio 7 (Bioinformatics Solutions, Inc., Ontario, Canada), and the peptide sequences were subjected to blastp (30) analysis for identification. Primers (HNDA_F [5′-TGCTGTCGCTGACGGC-3′] and HNDA_R [5′-TTGCTGAGCAGCACGAC-3′]) were designed on the basis of conserved regions exhibited in multiple-sequence alignment, generated by Clustalx v1.81 (31) using amidohydrolase gene sequences of various Burkholderia spp. (see Table S2 in the supplemental material). Using the primers, PCR was carried out in a 50-μl reaction volume using Phusion DNA polymerase (Thermo Fischer) in an MJ Mini Gradient Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA) with the following thermocycling conditions: 30 s at 98°C, followed by 30 cycles of 30 s at 98°C, 30 s at 55°C, and 10 s at 72°C. A final extension was performed at 72°C for 7 min. The resulting PCR product was sequenced as reported previously (27).

Enzyme assay.

Nonoxidative decarboxylase activity was qualitatively determined by UV-visible light spectral analysis as described previously (27) while the activity was quantitatively determined based on the formation of 2-naphthol, analyzed by high-pressure liquid chromatography (HPLC) using a methanol-water (50:50 vol/vol) isocratic solvent system with a flow rate of 1 ml/min (27). A standard curve of 2-naphthol created by HPLC under identical analytical conditions was used for quantitative estimation. One unit of enzyme activity is defined as the amount of enzyme required for the production of 1 μmol of product per min. The specific activity is expressed as units per milligram of protein.

For measurement of carboxylase activity, a standard reaction mixture containing recombinant protein (100 μg), 2-naphthol (20 mM), and NaHCO₃-NH₄HCO₃ (1.0 or 2.5 M) in a final volume of 1 ml of buffer A was prepared, and the reaction mixture was incubated for 60 min at 35°C. To analyze the reaction product, HPLC analysis was performed as described above.

Fosmid library construction, screening, and sequence analysis of amidohydrolase gene.

A genomic library of strain BC1 was prepared in E. coli using a CopyControl HTP fosmid library production kit (Epicentre, Madison, WI) according to the manufacturer's protocol. The resulting fosmid library was screened by PCR using HNDA_F and HNDA_R primers for clones harboring 2H1NA decarboxylase gene as described above. Fosmid DNA was isolated from the PCR-positive fosmid clones using the FosmidMAX DNA purification kit (Epicentre) and digested with EcoRI, HindIII, and SacII enzymes, and the DNA fragments (1 to 8 kb) were subcloned in pBluescript SK(−) vector. The colonies were rescreened by PCR using the same primer pair. The recombinant plasmids from the screened colonies were individually isolated and sequenced using M13 universal sequencing primers. The sequences were analyzed by BLAST analysis (version 2.2.12; National Center for Biotechnology Information [NCBI]), and the gaps between genes were bridged by using a conventional primer walking method.

Cloning, expression, and purification of recombinant proteins.

The primers Ex_HNDA_F (5′-CCGGAATTCATGACCGACCATCACCGTATC-3′) and Ex_HNDA_R (5′-CCCAAGCTTTTGTTGTGTTGTTGCGTCAG-3′) were designed (the EcoRI and HindIII restriction endonuclease recognition sites, respectively, are underlined) to amplify the complete 2H1NA decarboxylase gene (hndA) from genomic DNA of strain BC1. The amplified PCR product was digested with EcoRI and HindIII and ligated into similarly digested pET28a expression vector to form pET28a:HndA. The resulting plasmid was transformed into E. coli BL21(DE3) and plated on LB agar plates containing kanamycin. For the preparation of single amino acid substitution in HndA, pET28:HndA was subjected to whole-plasmid PCR with mutagenic primers (see Table S3 in the supplemental material) under following thermocycling conditions: 3 min at 98°C, followed by 16 cycles of 30 s at 98°C, 30 s at 55°C, and 3 min at 72°C, with a final extension at 72°C for 10 min using Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific). After digestion with DpnI for 2 h, the PCR product was transformed into electrocompetent E. coli Top10 and plated on LB agar plates containing kanamycin. Plasmids isolated from random clones were subjected to sequencing analysis to confirm the mutation at a specified location.

For recombinant enzyme expression and purification, E. coli BL21(DE3) cells harboring pET28a:HndA or its mutant derivatives (see Table S1 in the supplemental material) were grown in 500 ml of LB medium at 37°C with kanamycin to achieve an optical density at 600 nm of 0.5, followed by the addition of 0.5 mM IPTG (final concentration), and were grown further at 28°C for 3 h. The cultures were harvested by centrifugation (8,000 × g) and lysed in 10 ml of lysis buffer (50 mM NaHPO₄, 300 mM NaCl, 10 mM imidazole, and 10% glycerol) using a precooled French press (constant cell disruption system, One Shot model; United Kingdom) at 18,000 lb/in² for one cycle. After removal of the cell debris, the supernatant containing the His₆-tagged wild-type or mutant recombinant protein was purified by nickel-nitrilotriacetic acid (Ni²⁺-NTA)–agarose affinity chromatography using the purification buffers (wash buffer [50 mM NaHPO₄, 300 mM NaCl, 40 mM imidazole, 10% glycerol] and elution buffer [50 mM NaHPO₄, 300 mM NaCl, 250 mM imidazole, 10% glycerol]) according to the manufacturer's instructions (Qiagen). The purified protein fractions were pooled, dialyzed against buffer C, and analyzed by 12.5% SDS-PAGE. The dialyzed protein preparation was used in all biochemical studies.

Phylogenetic analysis.

Amino acid sequences of various proteins belonging to the amidohydrolase 1 and amidohydrolase 2 families were retrieved from the NCBI (see Table S4 in the supplemental material) and aligned, and a phylogenetic tree was constructed using the neighbor-joining algorithm, as implemented in ClustalX v1.81 (31). The tree was visualized using the program Tree Explorer v2.12, a stand-alone version of the same program implemented in MEGA 5 (32).

Gel filtration.

Native molecular mass of the decarboxylase was estimated by gel filtration chromatography using a P4000 PolySep GFC column (30 by 0.7 cm; Phenomenax, Torrance, CA), equilibrated with buffer A containing 200 mM NaCl. The flow rate used was 0.5 ml/min. Yeast alcohol dehydrogenase (150 kDa), conalbumin (75 kDa), ovalbumin (44 kDa), carbonic anhydrase (29 kDa), and RNase A (13.7 kDa) were used as standard proteins. Blue dextran (2,000 kDa) was used to calculate the void volume.

Biochemical studies.

All the enzyme kinetic analyses were done at 35°C and pH 7.5. For decarboxylation, the kinetic data were assessed using 1.5 μg of wild-type or mutant decarboxylase against 2H1NA over the concentration range of 0.05 to 0.5 mM. The maximum velocity (V_max) and the Michaelis constant (K_m) were determined from Lineweaver-Burk double-reciprocal plots using GraphPad Prism program (version 5.00 for Windows). The optimum temperature of the recombinant protein was determined over the range of 10 to 70°C, and the pH profile was determined at the optimal temperature determined as described above under standard conditions over the pH range of 4.0 to 9.5 using the following buffer systems (50 mM): citrate buffer (pH 4 to 6), sodium phosphate buffer (pH 6 to 8), and glycine-NaOH (pH 8.0 to 9.5). The effect of temperature on enzyme stability was determined by preincubating the enzyme at different temperatures (10 to 60°C) for 30 min and measuring the remaining activity under standard conditions. To study the effect of various metal ions and inhibitors on enzyme activity, purified 2H1NA decarboxylase preincubated with respective metal ions or inhibitors (1 or 5 mM) for 10 min at 4°C was used as an enzyme preparation. However, for metal chelators, viz., EDTA, 1,10-phenanthroline, 2,2′-bipyridyl, and 8-hydroxyquinoline-5-sulfonic acid (8-HQSA), enzyme was preincubated for 16 h.

For metal analysis, purified HndA (3 mg) was hydrolyzed by 65% ultrapure concentrated nitric acid (2 ml; Suprapure; Merck, Darmstadt, Germany) at 110°C for 1 h. The sample was diluted 10-fold by deionized double-distilled water, and the metal content was determined by using an atomic absorption spectrometer (iCE 3000 Series; Thermo Fischer Scientific).

Homology modeling and docking analyses.

A three-dimensional (3D) model of HndA was constructed using 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase (ACMSD) from Pseudomonas fluorescens (PDB accession no. 2HBV) (33) as a template employing Modeler 9v7 (34). The models were checked using Prochek, Verify3D, and VADAR (35–37). The NCBI PubChem database (http://pubchem.ncbi.nlm.nih.gov/) was used to obtain coordinates of the ligand 2H1NA. Preparation of protein and substrate files (pdbqt files) was performed using AutoDockTools-1.5.6 using default parameters (38). The grid box (with the dimensions 50 by 50 by 50 grid points) was generated using the AutoGrid4 program, keeping the metal ion coordinates (43.43 by −0.323 by 16.82) at the center. AutoDock4 was used to perform docking using genetic algorithm. Docked poses were analyzed using AutoDockTools-1.5.6 to get the best binding pose of 2H1NA with the lowest binding energy. The binding residues were identified and the schematic diagram of protein-ligand interaction was generated using LigPlot⁺ suite (version 1.4.5) (39).

Nucleotide sequence accession number.

The nucleotide sequence reported here has been deposited in the DDBJ/EMBL/GenBank database under accession number KU254672.

RESULTS

Partial purification and gene identification of 2H1NA decarboxylase.

2H1NA decarboxylase activity was previously reported to be strictly inducible in the presence of 2H1NA and a differentially expressing ∼32-kDa protein band was observed only in the cell extract of 2H1NA-grown cells compared to that of 2-naphthol-grown cells (27). For detailed characterization, 2H1NA decarboxylase was partially purified from crude cell extract of strain BC1 grown on 2H1NA using differential protein precipitation steps and hydrophobic interaction chromatography (Table 1). The purified enzyme preparation was found to be stable during the purification steps, carried out under aerobic conditions, indicating the oxygen-insensitive nature of the decarboxylase. The decarboxylase-active fractions from a phenyl-Sepharose column represented 19-fold purification (specific activity, 3.8 U mg⁻¹) with a yield of 16.8% and showed the presence of an ∼32-kDa band in SDS-PAGE, supporting our earlier observation (see Fig. S1 in the supplemental material). To confirm its identity, the ∼32-kDa protein was subjected to MALDI-TOF MS/MS analysis, where the generated peptide fragments showed strong sequence similarity to the uncharacterized amidohydrolase 2 proteins of various Burkholderia spp. in blastp analyses (see Table S5 in the supplemental material). Subsequently, a 220-bp PCR product was amplified (data not shown) from the genomic DNA of strain BC1 using the primers HNDA_F and HNDA_R, which on sequence analysis confirmed the results as stated above.

TABLE 1.

Purification summary of 2H1NA decarboxylase from Burkholderia sp. BC1

Step	Total protein (mg)	Total activity (U)	Sp acta (U/mg)	Purification (fold)	Yield (%)
Cell extract	180	36.3	0.2	1	100
Ammonium sulfate fractionation (30 to 50% saturation)	32	25.9	0.8	4	71.3
Phenyl-Sepharose 6 (Fast Flow) chromatography	1.6	6.1	3.8	19	16.8

^aSp act, specific activity.

Cloning and sequencing of the 2H1NA decarboxylase gene.

Screening of a genomic fosmid library of strain BC1 by PCR led to the identification of a subclone, which upon IPTG induction displayed 2H1NA decarboxylase activity, determined in the cell-free enzyme preparation. Complete sequence analysis of the subclone harboring a 4.2-kb EcoRI fragment revealed the presence of an amidohydrolase gene, designated hndA for hydroxynaphthoate decarboxylase. hndA, consisting of 951 nucleotides, encoded a polypeptide of 316 amino acids with a theoretical molecular mass of 34 kDa and pI of 5.59. Moreover, the CDD (Conserved Domain Database) and COG (Clusters of Orthologous Groups) analyses of HndA placed it in the amidohydrolase superfamily of the triosephosphate isomerase (TIM)-barrel fold protein (COG2159), which includes several nonoxidative decarboxylases, including 5-carboxyvanillate decarboxylase (5-CVD), 2,3-dihydroxybenzoate decarboxylase, and γ-resorcylate decarboxylase (γ-RSD) (4, 14, 15). HndA showed 71 to 97% identity with the biochemically uncharacterized metal-dependent hydrolase proteins of several Burkholderia species belonging to the amidohydrolase superfamily, listed in the NCBI database. However, among the biochemically well-characterized nonoxidative decarboxylases, HndA showed a modest identity of 27 and 24% with the γ-resorcylate decarboxylase (γ-RSD) of Rhizobium sp. strain MTP-10005 (15) and ACMSD of Pseudomonas fluorescens (10), respectively. Other genes in the 4.2-kb gene cluster include orf1, orf2, and dbpA, where the gene products showed 99 to 100% identity with a noncharacterized phenol degradation protein, a LysR-type regulator, and an ATP-dependent RNA helicase protein of Burkholderia multivorans, respectively. The genetic assembly of the 4.2-kb cluster is shown in Fig. 1A.

FIG 1.

(A) Gene organization of 4.2-kb EcoRI fragment showing gene designations. orf1, partial phenol degradation protein; hndA, 2H1NA decarboxylase; orf2, LysR-type-like regulator; dbpA, ATP-dependent RNA helicase. (B) Phylogenetic tree based on protein sequences from amidohydrolase 1 and amidohydrolase 2 family of proteins. Numbers at the nodes indicate the levels of bootstrap support based on neighbor-joining analysis of 100 resampled data sets. Bootstrap values below 50% are not shown. The scale bar represents 0.1 substitutions per nucleotide position. GenBank or PDB accession numbers are indicated within parentheses. Amidohydrolase 3 from Burkholderia sp. strain lig30 (KDB07616) was used as an outgroup. (C) Multiple-sequence alignment of protein sequences of the representative members of amidohydrolase 2 protein family. Metal coordinating residues are shaded.

Phylogenetic analysis of HndA.

A phylogenetic tree (Fig. 1B) constructed using multiple-sequence alignment of various proteins belonging to the amidohydrolase 1 and amidohydrolase 2 family positioned HndA within the amidohydrolase 2 family. With the exception of 4-oxalomesaconate hydratase (OMAH) from Sphingomonas paucimobilis SYK-6 (40), the other representative members belonging to this family are nonoxidative decarboxylases, viz., isoorotate decarboxylase (IDCase) from Neurospora crassa (12) and 5-carboxyvanillate decarboxylase (5-CVD) from Sphingomonas paucimobilis SYK-6 (4), apart from γ-RSD from Rhizobium sp. strain MTP-10005 (15) and ACMSD from Pseudomonas fluorescens (10). In addition, a number of uncharacterized metal-dependent hydrolases from Burkholderia also appeared to belong to this enzyme family (Fig. 1B).

Despite an overall low sequence homology among the biochemically characterized members of amidohydrolase 2 family proteins, the sequence alignment did display a strong residue conservation pattern for amino acids (His10, His12, His156, and Asp269 in HndA) that are responsible for the binding of the metal cofactor, which is crucial for enzyme catalysis (Fig. 1C). Apart from the results reported above, the alignment showed another conserved histidine residue (His204 in HndA) which was earlier reported to play a crucial role in enzyme catalysis for both ACMSD and γ-RSD (33, 41).

Overexpression and purification of recombinant HndA.

The recombinant 2H1NA decarboxylase was successfully overexpressed in E. coli BL21(DE3) with a 0.5 mM IPTG concentration and was purified by Ni²⁺-NTA chromatography (Fig. 2A). The purified recombinant enzyme migrated as a single band in the SDS-PAGE gel with an apparent subunit molecular mass of ∼38 kDa, while the molecular mass of the native recombinant enzyme on gel filtration was found to be 38.1 ± 0.5 kDa, suggesting the monomeric nature of the enzyme. Purified recombinant HndA catalyzed the decarboxylation of 2H1NA to 2-naphthol, as revealed by both spectral and HPLC analyses (Fig. 2B and C), with a specific activity of 9.0 U/mg of protein. Figure 2D shows the time-dependent transformation of 2H1NA to 2-naphthol by purified HndA over a period of 10 min.

FIG 2.

(A) SDS-PAGE analysis of overexpressed recombinant HndA protein. Lane 1, crude extract of E. coli BL21(DE3) carrying empty pET28a vector; lane 2, crude extracts of induced E. coli BL21(DE3) carrying pET28a:HndA; lane 3, purified recombinant HndA protein; lane M, molecular mass marker (Puregene). (B) Spectral changes during transformation of 2HINA by purified recombinant HndA protein. The sample and reference cuvettes contained 50 mM potassium phosphate buffer (pH 7.0) in a 1-ml volume. The sample cuvette also contained 220 nmol of 2H1NA. Spectra were recorded every 1 min after the addition of 10 μg of protein to both cuvettes. The up and down arrows indicate increasing and decreasing absorbances, respectively. (C) HPLC chromatogram showing transformation of 2H1NA to 2-naphthol by purified HndA in a reaction mixture (final volume, 1 ml) containing 0.5 mM 2H1NA and 5 μg of protein in buffer A incubated for 10 min at 35°C. UV-visible light spectra of 2H1NA and 2-naphthol are shown in insets. (D) Time-dependent transformation of 2H1NA to 2-naphthol by purified HndA. The concentrations of 2H1NA (○) and 2-naphthol (●) were determined by HPLC from the reaction mixtures (as described in panel C) during enzymatic transformation over 10 min.

Biochemical properties of recombinant HndA.

It was observed that HndA did not favor carboxylation of 2-naphthol under the conditions tested, and thus it appears that the enzyme catalyzes an irreversible reaction (decarboxylation). Again, HndA showed strict substrate specificity toward 2H1NA since its other structural isomers, 1H2NA and 3H2NA, could not be transformed. Also, it failed to decarboxylate mono- and dihydroxybenzoic acids, viz., 2-hydroxybenzoic acid (salicylic acid), 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid (gentisic acid), and 2,6-dihydroxybenzoic acid (γ-resorcylic acid). Similarly, HndA failed to transform phthalic acid, 1-naphthoic acid, and 2-naphthoic acid.

For 2H1NA decarboxylase, the values of K_m and V_max were determined to be 0.17 mM and 0.02 μmol/min, respectively. The k_cat/K_m value for 2H1NA was 47.05 mM⁻¹ s⁻¹. The optimum pH and temperature of the protein were found to be 7.5 and 35°C, respectively (see Fig. S2A and B in the supplemental material). The enzyme was found to be stable up to 45°C and retained 58% of initial activity when incubated at 50°C for 30 min. However, the enzyme completely lost its activity when incubated above 60°C (see Fig. S2C in the supplemental material).

The effect of various metal ions as well as inhibitors on enzyme activity is shown in Table S6 in the supplemental material. No significant change in enzyme activity was observed with majority of the metal ions, inhibitors, and metal ion chelators, individually incubated for 10 min. However, activity of the enzyme was inhibited by AgNO₃ and HgCl₂, as suggested for γ-RSD (15, 42). Nevertheless, a modest inhibition in HndA activity was observed when the enzyme was incubated for 16 h individually with metal chelators, including 8-hydroxy-quinoline-5-sulfonic acid, a zinc metal-specific inhibitor. This observation suggests the possible presence of a deeply embedded metal ion, inadequately accessible by metal chelators. Diethylpyrocarbonate, a histidine residue modifier, also showed a reasonable decrease in enzymatic activity, suggesting the presence of active-site histidine residues in HndA, as described earlier (27). To identify the metal center in the active site of HndA, atomic absorption spectroscopy analysis was performed that revealed the presence of zinc at 0.95 ± 0.1 mol per mol of protein. This result corroborated well with results determined for other nonoxidative decarboxylases belonging to the amidohydrolase superfamily that possess zinc as the transition metal center (33, 41).

Structural modeling and functional analysis of HndA mutants.

A 3D structural model of HndA showed the presence of a (β/α)₈ barrel fold with eight parallel β strands flanked by eight α helices on the outer face. The structural proximity of the conserved His10, His12, His156, and Asp269 residues in the 3D model of HndA was fully compatible with their putative role in forming a metal ion binding motif (Fig. 3A). In order to confirm the functional roles of these amino acid residues, site-directed mutants HndA^H10Q, HndA^H156Q, and HndA^D269V were constructed and expressed as soluble proteins (see Fig. S3 in the supplemental material), and their specific activities against 2H1NA were determined. HndA^H10Q and HndA^D269V showed no activity against 2H1NA, whereas HndA^H156Q retained only 7.23% (0.65 U/mg) of the wild-type decarboxylase activity. To analyze the role of the conserved histidine residue His204, the mutant protein HndA^H204Q was studied and showed a complete loss of enzymatic activity, suggesting a critical role of this residue in HndA-mediated catalysis.

FIG 3.

(A) Schematic representation of the structural model of HndA showing the enzyme active site. The inset shows the metal coordinating residues His10, His12, His156, and Asp269. (B) Surface topology of HndA showing the binding of 2H1NA within the catalytic pocket via electrostatic interaction with active-site residues Tyr272 and Arg33 based on docking analysis.

In order to determine other important amino acid residues responsible for substrate binding, docking analysis was performed using 2H1NA as a ligand. Docking analysis revealed the role of two important amino acid residues, Arg33 and Tyr272, which were found to interact with the carboxyl functional group of 2H1NA by hydrogen bonding (Fig. 3B). In addition to the carboxyl group, Tyr272 was also found to interact with the hydroxyl group of 2H1NA (Fig. 3B). To further assess their role in substrate binding, HndA^R33L and HndA^Y272F mutant proteins were generated (see Fig. S3 in the supplemental material), and subsequently their activities were tested against 2H1NA. The mutant enzymes, HndA^R33L and HndA^Y272F, showed specific activities of 4.1 and 2.8 U/mg of protein, respectively. Kinetic parameters determined for these mutants showed negligible change in K_m for HndA^H156Q but showed a clear increase in K_m for mutants HndA^R33L and HndA^Y272F, suggesting their role as substrate-binding residues. Also, the k_cat/K_m values for the mutants decreased by nearly 6- to 15-fold with respect to wild-type protein, indicating that all of these residues are essential for 2H1NA decarboxylation reaction (Table 2).

TABLE 2.

Kinetic constants of wild-type and mutant 2H1NA decarboxylases

Enzyme	Km (mM)	Vmax (μmol min−1)	kcat (s−1)	kcat/Km (mM−1 s−1)
HnD	0.17	0.02	7.99	47.27
HnDH10Q	NDa	ND	ND	ND
HnDH156Q	0.17	0.001	0.56	3.31
HnDD269V	ND	ND	ND	ND
HnDH204Q	ND	ND	ND	ND
HnDR33L	0.78	0.01	6.33	8.06
HnDY272F	0.31	0.007	3.02	9.71

^aND, not determined (product could not be detected).

DISCUSSION

Decarboxylation is ubiquitous in nature and is of fundamental biological importance. Among the different classes of decarboxylases, nonoxidative decarboxylases have received specific attention primarily because they catalyze transition metal-dependent decarboxylation without using molecular oxygen as a cosubstrate (1). Microorganisms expressing these enzymes not only play a significant role in biodegradation and/or bioremediation of soil, water, and sediment contaminated with lignin-related compounds and benzene derivatives of industrial origin but also act as biocatalysts in industrial biotransformation reactions (4, 15, 17).

Nonoxidative decarboxylases belonging to structurally distinct protein families that catalyze either reversible or irreversible reactions differ in their oxygen sensitivities (42). In aromatic acid metabolism, the presence of a variety of hydroxybenzoic acid nonoxidative decarboxylases has been detected, and some have been purified and characterized (2, 5, 14, 15, 42). To the best of our knowledge, the oxygen-insensitive 2H1NA nonoxidative decarboxylase described in the present study is the first bacterial enzyme belonging to the amidohydrolase superfamily that catalyzes an irreversible decarboxylation of a hydroxynaphthoic acid.

The amidohydrolase superfamily is comprised of functionally diverse enzymes that catalyze the cleavage of the C–N, C–C, C–O, C–Cl, C–S, or O–P bond of structurally distinct organic compounds (43–45). Generally, the members of the amidohydrolase superfamily share a signature for mono- or binuclear metal center embedded within the TIM-like barrel fold in the catalytic domain (43, 44). Within the amidohydrolase superfamily, members of the amidohydrolase 1 family catalyze hydrolytic reactions, while the amidohydrolase 2 family proteins are primarily involved in nonhydrolytic C-C bond cleavage (44). The gene encoding the 2H1NA decarboxylase (hndA) displayed similarities with the members of the amidohydrolase 2 family. In this family, ACMSD from Pseudomonas fluorescens is the first characterized member involved in 2-nitrobenzoic acid degradation pathway (10). Other members belonging to this family include 5CVD, γ-RSD, salicylic acid decarboxylase, OMAH, and Idcase (4, 15, 17, 40, 46). Since none of the enzymes belonging to the amidohydrolase 2 family possess hydrolase activity, Liu and Zhang (43) had proposed to rename this family the ACMSD-related protein family. Based on sequence similarity and phylogenetic analysis (Fig. 1), we propose HndA to be a new member of the ACMSD-related protein family.

Multiple-sequence alignment of ACMSD-related protein family members, including HndA, revealed a strict conservation pattern for key amino acid residues which act as important metal-binding protein ligands (43). For HndA, His10 and His12 constitute the conserved “HXH” metal-binding motif, whereas H156 and D269 are the other two endogenous metal-binding ligands (Fig. 3A). Enzyme inhibition by histidine residue-specific inhibitor diethylpyrocarbonate (DEPC) and site-directed mutagenesis studies revealed that the conserved histidine residues constitute the active-site protein ligands (Table 2). Interestingly, substitution of another conserved histidine residue (His204), not directly involved in metal binding, leads to the complete inactivation of the protein. This result is similar to that observed in ACMSD, where the corresponding residue (His228) was suggested to play the role of an acid-base catalyst involved in deprotonation of the metal-bound water, facilitating the decarboxylation of ACMS (47). On the other hand, docking analysis revealed that carboxyl and hydroxyl groups of 2H1NA are hydrogen bonded with Arg33/Tyr272 and Tyr272 of HndA, respectively. Interestingly, the importance of the arginine residue in substrate binding via carboxylate group has been suggested in ACMSD (33, 48). Similarly, the role of the active-site tyrosine residue in the binding of the hydroxyl group of lactic acid was studied in flavocytochrome b₂ or l-lactate dehydrogenase, and it was reported to play a role in converting lactic acid to pyruvic acid (49). The significance of Arg33 and Tyr 272 for efficient substrate binding in HndA was also confirmed by mutational analysis (Table 2).

Being a member of metallo-dependent hydrolase superfamily, HndA did not show any enhancement in activity when a set of metal ions were individually supplemented externally. Again, common divalent metal chelators had only mild inhibitory effects on this enzyme (see Table S6 in the supplemental material) even after prolonged incubation, suggesting a probable deeply buried metal center within the protein molecule. Modest inhibition by a zinc metal-specific inhibitor, 8-hydroxy-quinoloine-5-sulfonic acid (see Table S6 in the supplemental material), that suggested the possible presence of a zinc metal center within the enzyme was verified by atomic absorption spectroscopy. Additional biophysical investigations on HndA will provide further insights on the catalytic mechanism and structure-function relationships of this unique transition metal-dependent, oxygen-independent 2H1NA decarboxylase.