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Functional Characterization of 2-Hydroxy-1-naphthoic Acid Non-oxidative 1
Decarboxylase from Burkholderia sp. Strain BC1: A Novel Member of Amidohydrolase-2
2 Protein Family 3
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Piyali Pal Chowdhury, Soumik Basu, Arindam Dutta and Tapan K. Dutta# 6
Department of Microbiology, Bose Institute, Kolkata, India 7
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Running Title: Non-oxidative decarboxylase from Burkholderia sp. 9
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#Address correspondence to Tapan K. Dutta, [email protected] 12
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This manuscript contains supplementary data 18
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JB Accepted Manuscript Posted Online 11 April 2016J. Bacteriol. doi:10.1128/JB.00250-16Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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ABSTRACT 24
The gene encoding a non-oxidative decarboxylase capable of catalyzing the transformation of 25
2-hydroxy-1-naphthoic acid (2H1NA) to 2-naphthol was identified, recombinantly expressed 26
and purified to homogeneity. The putative gene sequence of the decarboxylase (hndA) 27
encodes a 316 amino acid protein (HndA) with a predicted molecular mass of 34 kDa. HndA 28
exhibited high identity with uncharacterized amidohydrolase-2 proteins of various 29
Burkholderia sp. whereas it showed a modest 27% identity with γ-resorcylate decarboxylase, 30
a well characterized non-oxidative decarboxylase belonging to the amidohydrolase 31
superfamily. Biochemically characterized HndA demonstrated strict substrate specificity 32
towards 2H1NA while inhibition studies with HndA indicated the presence of zinc as the 33
transition metal centre as confirmed by atomic absorption spectroscopy. A three-dimensional 34
structural model of HndA followed by docking analysis identified the conserved metal 35
coordinating and substrate binding residues, while their importance in catalysis was validated 36
by site-directed mutagenesis. 37
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IMPORTANCE 39
Microbial non-oxidative decarboxylases play a crucial role in the metabolism of a large array 40
of carboxy aromatic chemicals released into environment from a variety of natural and 41
anthropogenic sources. Among these, hydroxynaphthoic acids are usually encountered as 42
pathway intermediates in the bacterial degradation of polycyclic aromatic hydrocarbons. The 43
present study reveals biochemical and molecular characterization of a 2-hydroxy-1-naphthoic 44
acid non-oxidative decarboxylase involved in an alternative metabolic pathway which can be 45
classified as a member of the small repertoire of non-oxidative decarboxylases belonging to 46
the amidohydrolase-2 family of proteins. The strict substrate specificity and sequence 47
uniqueness makes it a novel member of the metallo-dependent hydrolase superfamily. 48
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INTRODUCTION 50
Decarboxylase is one of the most important classes of enzymes involved in a large variety of 51
catabolic and anabolic pathways. The majority of the decarboxylases utilize an organic 52
cofactor or a transition metal coupled with dioxygen to activate their substrates leading to the 53
removal of carbon dioxide (1). However, there is a small group of transition metal-dependent 54
decarboxylases that carry out decarboxylation of various aromatic acids in a non-oxidative 55
manner. These non-oxidative decarboxylases act on various lignin-derived compounds such 56
as 4-hydroxybenzoic acid (2) (carboxy)vanillic acid (3, 4), protocatechuic acid (5), ferulic 57
acid (6), p-coumaric acid (7) and estrogenic phthalate (8). Likewise, oxygen-independent 58
decarboxylases are also involved in 2-nitrobenzoic acid degradation pathway (9, 10), 59
tryptophan catabolic pathway (11) and thymidine salvage pathway (12). 60
Non-oxidative decarboxylases, in general, can broadly be classified into two major 61
groups depending on their oxygen sensitivity. Oxygen-sensitive decarboxylases, viz. 4-62
hydroxybenzoate decarboxylase (2), 3,4-dihydroxybenzoate decarboxylase (5) and indole-3-63
carboxylate decarboxylase (13) catalyze reversible reactions, both carboxylation and 64
decarboxylation. On the other hand, oxygen-insensitive decarboxylases such as 2,3-65
dihydroxybenzoate decarboxylase (14), 5-carboxyvanillate decarboxylase (4) and 4,5-66
dihydroxyphthalate decarboxylase (8) have been reported to catalyze the decarboxylation 67
reaction only. However, there are a few non-oxidative oxygen-insensitive decarboxylases, 68
viz. γ-resorcylate decarboxylase (15), vanillate/4-hydroxybenzoate decarboxylase (16) and 69
salicylate decarboxylase (17) that have been documented to catalyze reversible reactions. 70
Hydroxynaphthoates, such as 1-hydroxy-2-naphthoic acid (1H2NA), 2-hydroxy-1-71
naphthoic acid (2H1NA) and 3-hydroxy-2-naphthoic acid (3H2NA) are normally 72
encountered during the bacterial degradation of polycyclic aromatic hydrocarbons (PAHs), 73
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viz. phenanthrene, anthracene and pyrene, and are metabolized either through ring cleavage 74
(18-22) or by oxidative decarboxylation (23). In addition, decarboxylation of 1H2NA to 1-75
naphthol has been proposed, based on the identification of the later compound during 76
degradation of phenanthrene in a few bacteria (24-26). Similarly, decarboxylation of 77
phenanthrene-4,5-dicarboxylic acid to phenanthere-4-carboxylic acid has also been reported 78
in the pathway of pyrene degradation (19). However there is no documented report of any 79
non-oxidatively decarboxylated product, produced during the metabolism of 2H1NA or 80
3H2NA. Despite published reports of purification and characterization of several non-81
oxidative hydroxybenzoate decarboxylases (5, 14-16), there are no examples of any enzyme 82
catalyzing non-oxidative decarboxylation of any of the hydroxynaphthoic acid isomers. 83
Previously, we had reported a non-conventional degradation pathway of 2H1NA in 84
Burkholderia sp. BC1 describing 2-naphthol, gentisaldehyde and gentisic acid as pathway 85
intermediates (27). Moreover, presence of a strictly inducible non-oxidative decarboxylase 86
was also observed in the cell-free extract of 2H1NA-grown culture catalyzing the enzymatic 87
transformation of 2H1NA to 2-naphthol. In the present study, we describe a proteomic 88
approach-based gene cloning and functional characterization of non-oxidative 2H1NA 89
decarboxylase from Burkholderia sp. BC1. In addition, the roles of specific amino acid 90
residues responsible for substrate binding and enzyme catalysis have been elucidated. 91
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MATERIALS AND METHODS 93
Bacterial strains, plasmids and culture conditions. Strains and plasmids used in this study 94
are listed in Table S1 in supplemental material. Recombinant constructs in E. coli [XL1-Blue 95
and BL21(DE3)] were routinely grown and maintained in Luria-Bertani (LB) broth (per litre) 96
containing 10 g bactotryptone, 5 g yeast extract, and 10 g NaCl; pH 7.2 or on LB solid 97
medium (1.8 % w/v agar) at 37°C. Where appropriate, ampicillin (100 µg/ml), kanamycin 98
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(50 µg/ml), chloramphenicol (12.5 µg/ml), isopropyl-β-D-thiogalactopyranoside (IPTG) 99
(0.1 to 1 mM) or 5‐bromo‐4‐chloro‐3‐indolyl β‐D‐galactopyranoside (X‐gal) (20 µg/ml) 100
was added. For expression cloning, pET28a (Novagen, Madison, WI) served as the 101
expression vector. 102
Partial purification and gene identification of 2H1NA decarboxylase. Native 2H1NA 103
decarboxylase was purified from crude cell-free extract of BC1 cells grown for 16 h at 28°C 104
in four liters of MSM (20) containing 0.5 g/liter 2H1NA. Crude cell-free extract was prepared 105
as described previously (27), which was then fractionated by sequential protein precipitation 106
using ammonium sulphate. The 30-50% ammonium sulphate saturated fraction was 107
centrifuged at 12000×g for 30 min and the resulting pellet was dissolved in buffer A (50 mM 108
K2HPO4-KH2PO4 buffer, pH 7.0) and dialyzed against buffer B (50 mM K2HPO4-KH2PO4 109
buffer, pH 7.0 containing 0.8 M (NH4)2SO4). The dialyzed fraction was then loaded onto a 110
column (2.5 cm×10 cm), packed with Phenyl Sepharose 6 Fast Flow, pre-equilibrated with 111
buffer B. The column was washed with five column volumes of buffer B and then the 112
adsorbed proteins were eluted in steps using 10 column volumes of each of buffer A 113
containing different concentration of (NH4)2SO4 (0.8 ̶ 0.05 M). Finally, the column was 114
washed with two column volumes of buffer A. All purification steps were carried out at 4°C 115
or on ice under aerobic conditions. Fractions exhibiting 2H1NA decarboxylase activity were 116
combined and dialyzed against buffer C (50 mM K2HPO4-KH2PO4 buffer, pH 7.0, 10% 117
glycerol), concentrated by ultrafiltration (Millipore, Massachusetts, USA) and stored at -80°C 118
until further use. The purity of the protein fractions obtained after ammonium sulphate 119
precipitation and hydrophobic interaction chromatography was evaluated by 12.5% SDS-120
PAGE analysis followed by Coomassie blue staining in the presence of pre-stained protein 121
molecular mass markers (Puregene, Genetix, India) by standard techniques. Protein 122
quantification was done using the method of Bradford (28). 123
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For identification of the decarboxylase, tryptic digestion of the protein and subsequent 124
extraction of peptides from SDS-PAGE gel matrices were carried out by methods described 125
by Shevchenko et al. (29) followed by MALDI-TOF MS and MS/MS analyses using 126
AutoFlex II (Bruker Daltonics, Germany) MALDI-tandem time-of-flight (TOF/TOF) mass 127
spectrometer equipped with a pulsed N2 laser (λ = 337 nm, 50 Hz). The mass spectra were 128
analysed with Flex Analysis Software (version 2.4, Bruker, Daltonics). From the MS/MS 129
data, partial amino acid sequences of the peptides were determined using PEAKS studio 7 130
(Bioinformatics solutions, Ontario, Canada) and the peptide sequences were subjected to 131
blastp (30) analysis for identification. Primers [HNDA_F, 5′-TGCTGTCGCTGACGGC-3′ and 132
HNDA_R, 5′-TTGCTGAGCAGCACGAC-3′] were designed on the basis of conserved regions 133
exhibited in multiple sequence alignment, generated by Clustalx v1.81 (31) using 134
amidohydrolase gene sequences of various Burkholderia sp. (see Table S2 in supplemental 135
material). Using the primers, PCR was carried out in a 50-µl reaction volume using phusion 136
DNA polymerase (Thermo Fischer) in a MJ Mini Gradient Thermal Cycler (Bio-Rad 137
Laboratories, Inc., Hercules, CA, USA) with the following thermo-cycling conditions: 30 s at 138
98°C followed by 30 cycles of 30 s at 98°C, 30 s at 55°C and 10 s at 72°C. Final extension 139
was performed at 72°C for 7:00 min. The resulting PCR product was sequenced as reported 140
previously (27) 141
Enzyme assay. Non-oxidative decarboxylase activity was qualitatively determined by UV-142
visible spectral analysis as described previously (27) while the activity was quantitatively 143
determined based on the formation of 2-naphthol, analyzed by HPLC using a methanol/water 144
(50:50 v/v) isocratic solvent system with a flow rate of 1 ml/min (27). A standard curve of 2-145
naphthol created by HPLC under identical analytical conditions was used for quantitative 146
estimation. One unit of enzyme activity is defined as the amount of enzyme required for the 147
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production of 1 μmol of product per min. Specific activity is expressed as units per mg of 148
protein. 149
For measurement of carboxylase activity, a standard reaction mixture containing 150
recombinant protein (100 μg), 2-naphthol (20 mM) and NaHCO3/NH4HCO3 (1.0 or 2.5 M) in 151
a final volume of 1 ml of buffer A was prepared and the reaction mixture was incubated for 152
60 min at 35°C. To analyze the reaction product, HPLC analysis was performed as described 153
above. 154
Fosmid library construction, screening and sequence analysis of amidohydrolase gene. 155
A genomic library of strain BC1 was prepared in E. coli using CopyControl™ HTP Fosmid 156
Library Production Kit (Epicentre, Madison, Wisconsin) according to the manufacturer’s 157
protocol (Epicentre, Madison, Wisconsin). The resulting fosmid library was screened by 158
PCR using HNDA_F and HNDA_R primers for clones harbouring 2H1NA decarboxylase 159
gene as described above. Fosmid DNA was isolated from the PCR positive fosmid clones 160
using the FosmidMAX™ DNA Purification Kit (Epicentre, Madison, Wisconsin), digested 161
with EcoRI, HindIII and SacII enzymes and the DNA fragments (1-8 kb) were subcloned in 162
pBluescript SK(-) vector. The colonies were rescreened by PCR using the same primer pair. 163
The recombinant plasmids from the screened colonies were individually isolated and 164
sequenced using M13 universal sequencing primers. The sequences were analyzed by 165
BLAST analysis (version 2.2.12, National Center for Biotechnology Information) and the 166
gaps between genes were bridged by conventional primer walking method. 167
Cloning, expression and purification of recombinant proteins. Primers, Ex_HNDA_F [5′-168
CCGGAATTCATGACCGACCATCACCGTATC-3′] and Ex_HNDA_R [5′-169
CCCAAGCTTTTGTTGTGTTGTTGCGTCAG-3′] were designed (restriction endonuclease 170
recognition sites are underlined for EcoRI and HinDIII respectively) to amplify the complete 171
2H1NA decarboxylase gene (hndA) from genomic DNA of strain BC1. The amplified PCR 172
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product was digested with EcoRI and HinDIII and ligated into similarly digested pET28a 173
expression vector to form pET28a:HndA. The resulting plasmid was transformed into E. coli 174
BL21(DE3) and plated on LB-agar plates containing kanamycin. For the preparation of single 175
amino acid substitution in HndA, pET28:HndA was subjected to whole plasmid PCR with 176
mutagenic primers (see Table S3 in Supplemental material) under following thermo-cycling 177
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 178
72°C with final extension at 72°C for 10 min using phusion high-fidelity DNA polymerase 179
(Thermo Fischer Scientific). After digestion with DpnI for 2 h, the PCR product was 180
transformed into electrocompetant E. coli Top10 and plated on LB-agar plates containing 181
kanamycin. Plasmids isolated from random clones were subjected to sequencing analysis to 182
confirm the mutation at specified location. 183
For recombinant enzyme expression and purification, E. coli BL21(DE3) cells harbouring 184
pET28a:HndA or its mutant derivatives (see Table S1 in Supplemental material) were grown 185
in 500 ml LB medium at 37°C with kanamycin to achieve an OD600 of 0.5 followed by the 186
addition of 0.5 mM IPTG (final concentration) and grown further at 28°C for 3 h. The 187
cultures were harvested by centrifugation (8000×g) and lysed in 10 ml of lysis buffer (50 mM 188
NaHPO4, 300 mM NaCl, 10 mM imidazole and 10 % glycerol) using a pre-cooled French 189
press (Constant Cell Disruption System, One Shot model, United Kingdom) at 18 Kpsi for 190
one cycle. After removal of the cell debris, the supernatant containing the His6-tagged wild 191
type or mutant recombinant protein was purified by Ni2+-NTA agarose affinity 192
chromatography using the purification buffers [wash buffer (50 mM NaHPO4, 300 mM NaCl, 193
40 mM imidazole and 10% glycerol) and elution buffer (50 mM NaHPO4, 300 mM NaCl, 194
250 mM imidazole and 10% glycerol)] according to the manufacturer’s instructions (Qiagen). 195
The purified protein fractions were pooled, dialyzed against buffer C and analyzed by 12.5% 196
SDS-PAGE. The dialyzed protein preparation was used in all biochemical studies. 197
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Phylogenetic analysis. The amino acid sequences of various proteins belonging to the 198
amidohydrolase-1 and amidohydrolase-2 families were retrieved from NCBI (see Table S4 in 199
Supplemental material), aligned and a phylogenetic tree was constructed using the neighbour-200
joining algorithm as implemented in ClustalX v1.81 (31). The tree was visualized using the 201
program Tree Explorer v2.12, a stand-alone version of the same program implemented in 202
MEGA 5 (32). 203
Gel filtration. Native molecular mass of the decarboxylase was estimated by gel filtration 204
chromatography using a P4000 PolySep GFC column (30×0.7 cm, Phenomenax, Torrance, 205
CA), equilibrated with buffer A containing 200 mM NaCl. Flow rate used was 0.5 ml/min. 206
Yeast alcohol dehydrogenase (150 kDa), conalbumin (75 kDa), ovalbumin (44 kDa), 207
carbonic anhydrase (29 kDa) and ribonuclease A (13.7 kDa) were used as standard proteins. 208
Blue dextran (2000 kDa) was used to calculate the void volume. 209
Biochemical studies. All the enzyme kinetic analyses were done at 35°C and pH 7.5. For 210
decarboxylation, the kinetic data were assessed using 1.5 μg of wild type or mutant 211
decarboxylase against 2H1NA over the concentration range of 0.05 to 0.5 mM. The 212
maximum velocity (Vmax) and the Michaelis constant (Km) were determined by Lineweaver-213
Burk double reciprocal plots using GRAPHPAD-PRISM program (version 5.00 for 214
Windows). The optimum temperature of the recombinant protein was determined over the 215
range of 10 to 70°C and the pH profile was determined over the pH range of 4.0-9.5 using the 216
following buffer systems (50 mM): citrate buffer (pH 4-6), sodium phosphate buffer (pH 6-217
8), and glycine-NaOH (pH 8.0-9.5) at the optimal temperature determined above under 218
standard conditions. The effect of temperature on enzyme stability was determined by 219
preincubating the enzyme at different temperatures (10-60°C) for 30 min and measuring the 220
remaining activity under standard conditions. To study the effect of various metal ions and 221
inhibitors on enzyme activity, purified 2H1NA decarboxylase preincubated with respective 222
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metal ions or inhibitors (1 or 5 mM) for 10 min at 4°C, was used as enzyme preparation. 223
However, for metal chelators, viz. EDTA, 1,10-phenanthroline, 2,2′-bipyridyl and 8-224
hydroxyquinoline-5-sulphonic acid (8-HQSA), enzyme was preincubated for 16h. 225
For metal analysis, purified HndA (3 mg) was hydrolyzed by 65% ultrapure concentrated 226
nitric acid (2 ml) (Suprapure, Merck, Darmstadt, Germany) at 110°C for 1 h. The sample was 227
diluted 10-fold by deionized double distilled water, and the metal content was determined by 228
atomic absorption spectrometer (iCE 3000 Series, Thermo Fischer Scientific). 229
Homology modeling and docking analyses. A three-dimensional model of HndA was 230
constructed using 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase (ACMSD) 231
from Pseudomonas fluorescens (PDB ID: 2HBV) (33) as template employing Modeller 9v7 232
(34). The models were checked using Prochek, Verify3D and VADAR (35-37). The NCBI 233
PubChem database (http://pubchem.ncbi.nlm. nih.gov/) was used to obtain co-ordinates of the 234
ligand 2H1NA. Preparation of protein and substrate files (pdbqt files) was performed using 235
AutoDockTools-1.5.6 using default parameters (38). The grid box with dimensions 236
50 × 50 × 50 grid points was generated using AutoGrid4 program keeping the metal ion 237
coordinates (43.43 × -0.323 × 16.82) at the centre. AutoDock4 was used to perform docking 238
using genetic algorithm. Docked poses were analyzed using AutoDockTools-1.5.6 to get the 239
best binding pose of 2H1NA with the lowest binding energy. The binding residues were 240
identified and the schematic diagram of protein-ligand interaction was generated using 241
LigPlot+ suite (version 1.4.5) (39). 242
Nucleotide sequence accession numbers. The nucleotide sequence reported in this paper has 243
been deposited in the DDBJ/EMBL/GenBank database under the accession number 244
KU254672. 245
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RESULTS 248
Partial purification and gene identification of 2H1NA decarboxylase. 2H1NA 249
decarboxylase activity was previously reported to be strictly inducible in the presence of 250
2H1NA and a differentially expressing ~32 kDa protein band was observed only in the cell-251
free extract of 2H1NA-grown cells when compared to that of 2–naphthol-grown cells (27). 252
For detailed characterization, 2H1NA decarboxylase was partially purified from crude cell-253
free extract of strain BC1 grown on 2H1NA using differential protein precipitation steps and 254
hydrophobic interaction chromatography (Table 1). The purified enzyme preparation was 255
found to be stable during the purification steps, carried out under aerobic conditions 256
indicating oxygen insensitive nature of the decarboxylase. The decarboxylase-active fractions 257
from Phenyl Sepharose column represented 19-fold purification (specific activity 3.8 U mg-1) 258
with a yield of 16.8% and showed the presence of a ~32 kDa band in SDS-PAGE supporting 259
our earlier observation (see Fig. S1 in Supplemental material). 260
To confirm its identity, the ~32 kDa protein was subjected to MALDI-TOF MS/MS 261
analysis where the generated peptide fragments showed strong sequence similarity with the 262
uncharacterized amidohydrolase-2 proteins of various Burkholderia sp. in Blastp analyses 263
(see Table S5 in Supplemental material). Subsequently, a 220 bp PCR product was amplified 264
(data not shown) from the genomic DNA of strain BC1 using the primers, HNDA_F and 265
HNDA_R, which on sequence analysis confirmed the results as stated above. 266
Cloning and sequencing of the 2H1NA decarboxylase gene. Screening of a genomic 267
fosmid library of strain BC1 by PCR led to the identification of a subclone, which upon IPTG 268
induction displayed 2H1NA decarboxylase activity, determined in the cell-free enzyme 269
preparation. Complete sequence analysis of the subclone harbouring a 4.2 kb EcoRI fragment 270
revealed the presence of the amidohydrolase gene designated as hndA for hydroxynaphthoate 271
decarboxylase. The decarboxylase, hndA, consisting of 951 nucleotides, encoded a 272
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polypeptide of 316 amino acids with a theoretical molecular weight of 34 kDa and pI of 5.59. 273
Moreover, the CDD (Conserved Domain Database) and COG (Clusters of Orthologous 274
Groups) analyses of HndA placed it in the amidohydrolase superfamily of the TIM-barrel 275
fold protein (COG2159), which includes several non-oxidative decarboxylases, including 5-276
carboxyvanillate decarboxylase (5-CVD), 2,3-dihydroxybenzoate decarboxylase and γ-277
resorcylate decarboxylase (γ-RSD) (4, 14, 15). HndA showed 71-97% identity with the 278
biochemically uncharacterized metal-dependent hydrolase proteins of several Burkholderia 279
sp. belonging to the amidohydrolase superfamily, listed in the NCBI database. However, 280
among the biochemically well-characterized non-oxidative decarboxylases, HndA showed a 281
modest identity of 27 and 24% with the γ-resorcylate decarboxylase (γ-RSD) of Rhizobium 282
sp. MTP-10005 (15) and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase 283
(ACMSD) of Pseudomonas fluorescence (10), respectively. Other genes in the 4.2 kb gene 284
cluster includes orf1, orf2 and dbpA where the gene products showed 99-100% identity with 285
a non-characterized phenol degradation protein, a LysR-type regulator and an ATP-286
dependent RNA helicase protein of Burkholderia multivorans respectively. The genetic 287
assembly of the 4.2 kb cluster is shown in Fig. 1A. 288
Phylogenetic analysis of HndA. A phylogenetic tree (Fig. 1B) constructed using multiple 289
sequence alignment of various proteins belonging to the amidohydrolase-1 and 290
amidohydrolase-2 family positioned HndA within the amidohydrolase-2 family. With the 291
exception of 4-oxalomesaconate hydratase (OMAH) from Sphingomonas paucimobilis SYK-292
6 (40), the other representative members belonging to this family are non-oxidative 293
decarboxylases, viz. isoorotate decarboxylase (IDCase) from Neurospora crassa (12) and 5-294
carboxyvanillate decarboxylase (5-CVD) from Sphingomonas paucimobilis SYK-6 (4) apart 295
from γ-RSD from Rhizobium sp. MTP-10005 (15) and ACMSD from Pseudomonas 296
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fluorescence (10). In addition, a number of uncharacterized metal-dependent hydrolases from 297
Burkholderia also appeared to belong to this enzyme family (Fig. 1B). 298
Despite an overall low sequence homology among the biochemically characterized 299
members of amidohydrolase-2 family proteins, the sequence alignment did display strong 300
residue conservation pattern for amino acids (His10, His12, His156 and Asp269 in HndA) 301
that are responsible for the binding of the metal cofactor, crucial for enzyme catalysis (Fig. 302
1C). Apart from above, the alignment showed another conserved histidine residue (His204 in 303
HndA) which was earlier reported to play a crucial role in enzyme catalysis for both ACMSD 304
and γ-RSD (33, 41). 305
Overexpression and purification of recombinant of HndA. The recombinant 2H1NA 306
decarboxylase was successfully overexpressed in E. coli BL21(DE3) under 0.5 mM IPTG 307
concentration and was purified by Ni2+-NTA chromatography (Fig. 2A). The purified 308
recombinant enzyme migrated as a single band in the SDS-PAGE gel with an apparent 309
subunit molecular weight of ~38 kDa. While the molecular weight of the native recombinant 310
enzyme on gel filtration was found to be 38.1±0.5 kDa, suggesting the monomeric nature of 311
the enzyme. Purified recombinant HndA catalyzed the decarboxylation of 2H1NA to 2-312
naphthol as revealed by both spectral and HPLC analyses (Fig. 2B, 2C) with a specific 313
activity of 9.0 U/mg of protein. Fig. 2D shows the time-dependent transformation of 2H1NA 314
to 2-naphthol by purified HndA over a period of 10 minutes. 315
Biochemical properties of recombinant HndA. It was observed that HndA did not favour 316
carboxylation of 2-naphthol under the conditions tested and thus it appears that the enzyme 317
catalyzes an irreversible reaction (decarboxylation). Again, HndA showed strict substrate 318
specificity towards 2H1NA since its other structural isomers, 1H2NA and 3H2NA, could not 319
be transformed. Also, it failed to decarboxylate mono- and dihydroxybenzoic acids, viz. 2-320
hydroxybenzoic acid (salicylic acid), 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2,3-321
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dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid (gentisic acid) 322
and 2,6-dihydroxybenzoic acid (γ-resorcylic acid). Similarly, HndA failed to transform 323
phthalic acid, 1-naphthoic acid and 2-naphthoic acid. 324
For 2H1NA decarboxylase, the values of Km and Vmax were determined to be 0.17 mM and 325
0.02 μmol/min, respectively. The kcat/Km value for 2H1NA was 47.05 mM-1 s-1. The optimum 326
pH and temperature of the protein was found to be 7.5 and 35°C respectively (see Fig. S2A 327
and S2B in Supplemental material). The enzyme was found to be stable up to 45°C and 328
retained 58% of initial activity when incubated at 50°C for 30 min. However, the enzyme 329
completely lost its activity when incubated above 60°C (see Fig. S2C in Supplemental 330
material). 331
The effect of various metal ions as well as inhibitors on enzyme activity is shown in 332
supplementary Table S6. No significant change in enzyme activity was observed with 333
majority of the metal ions, inhibitors and metal ion chelators, individually incubated for 10 334
min. However, activity of the enzyme was inhibited by AgNO3 and HgCl2, as suggested for 335
γ-RSD (15, 42). Nevertheless, a modest inhibition in HndA activity was observed when the 336
enzyme was incubated for 16 h individually with metal chelators including 8-hydroxy-337
quinoline-5-sulfonic acid, a zinc metal specific inhibitor. This observation suggests the 338
possible presence of a deeply embedded metal ion, inadequately accessible by metal 339
chelators. Diethylpyrocarbonate, a histidine residue modifier also showed a reasonable 340
decrease in enzymatic activity suggesting the presence of active site histidine residues in 341
HndA as described earlier (28). To identify the metal centre in the active site of HndA, 342
atomic absorption spectroscopy analysis was performed which revealed the presence of zinc 343
at 0.95±0.1 mol per mol of protein. This result corroborated well with other non-oxidative 344
decarboxylases belonging to the amidohydrolase superfamily that possess zinc as the 345
transition metal centre (33, 41). 346
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Structural modelling and functional analysis of HndA mutants. A three-dimentional 347
(3D) structural model of HndA showed the presence of a (β/α)8 barrel fold with eight parallel 348
β strands flanked by eight α helices on the outer face. The structural proximity of the 349
conserved His10, His12, His156 and Asp269 residues in the 3D model of HndA was fully 350
compatible with their putative role in forming a metal ion binding motif (Fig. 3A). In order to 351
confirm the functional roles of these amino acid residues, site-directed mutants HndAH10Q, 352
HndAH156Q and HndAD269V were constructed, expressed as soluble proteins (see Fig. S3 in 353
Supplemental material) and their specific activity against 2H1NA were determined. 354
HndAH10Q and HndAD269V showed no activity against 2H1NA whereas HndAH156Q retained 355
only 7.23% (0.65 U/mg) of the wild type decarboxylase activity. To analyse the role of the 356
conserved histidine residue His204, the mutant protein HndAH204Q was studied which showed 357
a complete loss of enzymatic activity, suggesting a critical role of this residue in HndA-358
mediated catalysis. 359
In order to determine other important amino acid residues responsible for substrate 360
binding, docking analysis was performed using 2H1NA as a ligand. Docking analysis 361
revealed the role of two important amino acid residues, Arg33 and Tyr272, which were found 362
to interact with the carboxyl functional group of 2H1NA by hydrogen bonding (Fig. 3B). In 363
addition to the carboxyl group, Tyr272 was also found to interact with the hydroxyl group of 364
2H1NA (Fig. 3B). To further assess their role in substrate binding, HndAR33L and HndAY272F 365
mutant proteins were generated (see Fig. S3 in Supplemental material) and subsequently their 366
activities were tested against 2H1NA. The mutant enzymes, HndAR33L and HndAY272F, 367
showed specific activities of 4.1 and 2.8 U/mg of protein respectively. Kinetic parameters 368
determined for these mutants showed negligible change in Km for HndAH156Q but showed a 369
clear increase in Km for mutants HndAR33L and HndAY272F, suggesting their role as substrate 370
binding residues. Also, the kcat/Km values for the mutants decreased by nearly six to fifteen-371
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fold with respect to wild type protein indicating that all of these residues are essential for 372
2H1NA decarboxylation reaction (Table 2). 373
374
DISCUSSION 375
Decarboxylation is ubiquitous in nature and is of fundamental biological importance. Among 376
the different classes of decarboxylases, nonoxidative decarboxylases have received specific 377
attention primarily because they catalyze transition metal-dependent decarboxylation without 378
using molecular oxygen as a cosubstrate (1). Microorganisms expressing these enzymes not 379
only play a significant role in biodegradation and/or bioremediation of soil, water and 380
sediment-contaminated with lignin-related compounds and benzene-derivatives of industrial 381
origin but also act as biocatalysts in industrial biotransformation reactions (4, 15, 17). 382
Non-oxidative decarboxylases belonging to structurally distinct protein families differ in 383
their oxygen sensitivity that catalyze either reversible or irreversible reactions (42). In 384
aromatic acid metabolism, presence of a variety of hydroxybenzoic acid non-oxidative 385
decarboxylases has been detected and some have been purified and characterized (2, 5, 14, 386
15, 42). To the best of our knowledge, the oxygen insensitive 2H1NA non-oxidative 387
decarboxylase described in this study is the first bacterial enzyme belonging to the 388
amidohydrolase superfamily that catalyzes an irreversible decarboxylation of a 389
hydroxynaphthoic acid. 390
The amidohydrolase superfamily is comprised of functionally diverse enzymes that 391
catalyze the cleavage of C–N, C–C, C–O, C–Cl, C–S or O–P bond of structurally distinct 392
organic compounds (43-45). Generally, members of amidohydrolase superfamily share a 393
signature for mono- or binuclear metal centre embedded within the triosephosphate isomerase 394
(TIM)-like barrel fold in the catalytic domain (43, 44). Within the amidohydrolase 395
superfamily, members of the amidohydrolase-1 family catalyze hydrolytic reactions while the 396
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amidohydrolase-2 family proteins are primarily involved in non-hydrolytic C-C bond 397
cleavage (44). The gene encoding the 2H1NA decarboxylase (hndA) displayed similarities 398
with the members of the amidohydrolase-2 family. In this family, ACMSD from 399
Pseudomonas fluorescence is the first characterized member involved in 2-nitrobenzoic acid 400
degradation pathway (10). Other members belonging to this family include 5CVD, γ-RSD, 401
salicylic acid decarboxylase, OMAH and Idcase (4, 15, 17, 40, 46). Since none of the 402
enzymes belonging to amidohydrolase-2 family possess hydrolase activity, Liu & Zhang (43) 403
had proposed to rename this family as ACMSD-related protein family. Based on sequence 404
similarity and phylogenetic analysis (Fig. 1), we propose HndA to be a new member of 405
ACMSD-related protein family. 406
Multiple sequence alignment of ACMSD-related protein family members, including 407
HndA, revealed strict conservation pattern for key amino acid residues which act as important 408
metal binding protein ligands (43). For HndA, His10 and His12 constitute the conserved 409
“HxH” metal binding motif whereas H156 and D269 are the other two endogenous metal 410
binding ligands (Fig. 3A). Enzyme inhibition by histidine residue specific inhibitor 411
diethylpyrocarbonate (DEPC) and site directed mutagenesis studies revealed that the 412
conserved histidine residues constitute the active site protein ligands (Table 2). Interestingly, 413
substitution of another conserved histidine residue (His204), not directly involved in metal 414
binding, leads to the complete inactivation of the protein. This result is similar to that 415
observed in ACMSD where the corresponding residue (His228) was suggested to play the 416
role of an acid-base catalyst involved in deprotonation of the metal-bound water facilitating 417
the decarboxylation of ACMS (47). 418
On the other hand, docking analysis revealed that carboxyl and hydroxyl groups of 419
2H1NA are hydrogen bonded with Arg33/Tyr272 and Tyr272 of HndA, respectively. 420
Interestingly, the importance of arginine residue in substrate binding via carboxylate group 421
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has been suggested in ACMSD (33, 48). Similarly, the role of the active site tyrosine residue 422
in the binding of the hydroxyl group of lactic acid was studied in flavocytochromeb2 or L-423
lactate dehydrogenase and it was reported to play a role in converting lactic acid to pyruvic 424
acid (49). The significance of Arg33 and Tyr 272 for efficient substrate binding in HndA was 425
also confirmed by mutational analysis (Table 2). 426
Being a member of metallo-dependent hydrolase superfamily, HndA did not show any 427
enhancement in activity when a set of metal ions were individually supplemented externally. 428
Again, common divalent metal chelators had only mild inhibitory effects on this enzyme (see 429
Table S6 in supplemental material) even after prolonged incubation, suggesting a probable 430
deeply buried metal centre within the protein molecule. Modest inhibition by a zinc metal-431
specific inhibitor, 8-hydroxy-quinoloine-5-sulphonic acid (see Table S6 in supplemental 432
material) that suggested the possible presence of a zinc metal centre within the enzyme was 433
verified by atomic absorption spectroscopy. Additional biophysical investigations on HndA 434
will provide further insights on the catalytic mechanism and structure–function relationships 435
of this unique transition metal-dependent, oxygen-independent 2H1NA decarboxylase. 436
437
ACKNOWLEDGMENTS 438
Authors acknowledge Gautam Basu for editing the manuscript. Financial support for this 439
work was provided by Bose Institute, Kolkata, India. P.P.C. was supported with fellowships 440
from the Council of Scientific and Industrial Research, Government of India while S.B. and 441
A.D were supported with fellowships from Bose Institute. 442
443
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592
FIGURE LEGEND 593
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FIG 1 (A) Gene organization of 4.2 kb EcoRI fragment showing gene designations. orf1, 594
partial phenol degradation protein; hndA, 2H1NA decarboxylase; orf2, LysR-type like 595
regulator; dbpA, ATP-dependent RNA helicase. (B) Phylogenetic tree based on protein 596
sequences from amidohydrolase-1 and amidohydrolase-2 family of proteins. Numbers at 597
nodes indicate levels of bootstrap support based on neighbour joining analysis of 100 598
resampled data sets. Bootstrap values below 50% are not shown. Bar represents 0.1 599
substitutions per nucleotide position. GenBank or PDB accession numbers are indicated 600
within parentheses. Amidohydrolase-3 from Burkholderia sp. lig30 (KDB07616) was used as 601
an outgroup. (C) Multiple sequence alignment of protein sequences of the representative 602
members of amidohydrolase-2 protein family. Metal coordinating residues are shaded. 603
604
FIG 2 (A) SDS PAGE analysis of overexpressed recombinant HndA protein. Lane 1, crude 605
extract of E. coli BL21(DE3) carrying empty pET28a vector; lane 2, crude extracts of 606
induced E. coli BL21(DE3) carrying pET28a:HndA; lane 3, purified recombinant HndA 607
protein and lane M, molecular weight marker (Puregene, Genetix, India). (B) Spectral 608
changes during transformation of 2HINA by purified recombinant HndA protein. The sample 609
and reference cuvettes contained 50 mM potassium phosphate buffer (pH 7.0) in 1-ml 610
volume. The sample cuvette also contained 220 nmol 2H1NA. Spectra were recorded every 611
one minute after the addition of 10 µg protein to both cuvettes. The up and down arrows 612
indicate increasing and decreasing absorbance, respectively. (C) HPLC chromatogram 613
showing transformation of 2H1NA to 2-naphthol by purified HndA in a reaction mixture 614
(final volume, 1 ml) containing 0.5 mM 2H1NA and 5 µg protein in buffer A incubated for 615
10 min at 35°C. UV-visible spectra of 2H1NA and 2-naphthol are shown in insets. (D) Time-616
dependent transformation of 2H1NA to 2-naphthol by purified HndA. Concentration of 617
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2H1NA (○) and 2-naphthol (●) were determined by HPLC from the reaction mixtures (as 618
described in panel C) during enzymatic transformation over 10 minutes. 619
620
FIG 3 (A) Schematic representation of the structural model of HndA showing the enzyme 621
active site. Inset shows the metal coordinating residues His10, His12, His156 and Asp269. 622
(B) Surface topology of HndA showing the binding of 2H1NA within the catalytic pocket via 623
electrostatic interaction with active site residues Tyr272 and Arg33 based on docking 624
analysis. 625
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TABLE 1 Purification summary of 2H1NA decarboxylase from Burkholderia sp. BC1
Step
Total
protein
(mg)
Total
activity
(U)
Specific
activity
(U/mg)
Purification
(fold)
Yield
(%)
Cell-free extract 180 36.3 0.2 1 100
Ammonium sulphate
fractionation (30-50%
saturation)
32
25.9
0.8
4
71.3
Phenyl-sepharose 6 (Fast
Flow) 1.6 6.1 3.8 19 16.8
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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 ND 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
ND, Not determined (as product could not be detected).
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