Identification and Characterization of a Novel Gentisate 1,2-Dioxygenase Gene from a Halophilic Martelella Strain

Halophilic Martelella strain AD-3, isolated from highly saline petroleum-contaminated soil, can efficiently degrade polycyclic aromatic hydrocarbons (PAHs), such as phenanthrene and anthracene, in 3–5% salinity. Gentisic acid is a key intermediate in the microbial degradation of PAH compounds. However, there is little information on PAH degradation by moderately halophilic bacteria. In this study, a 1,077-bp long gene encoding gentisate 1,2-dioxygenase (GDO) from a halophilic Martelella strain AD-3 was cloned, sequenced, and expressed in Escherichia coli. The recombinant enzyme GDO was purified and characterized in detail. By using the 18O isotope experiment and LC-MS analysis, the sources of the two oxygen atoms added onto maleylpyruvate were identified as H2O and O2, respectively. The Km and kcat values for gentisic acid were determined to be 26.64 μM and 161.29 s−1, respectively. In addition, optimal GDO activity was observed at 30 °C, pH 7.0, and at 12% salinity. Site-directed mutagenesis demonstrated the importance of four highly conserved His residues at positions 155, 157, 167, and 169 for enzyme activity. This finding provides new insights into mechanism and variety of gentisate 1,2-dioxygenase for PAH degradation in high saline conditions.

Scientific RepoRts | 5:14307 | DOi: 10.1038/srep14307 Martelella sp. strain AD-3, a moderate halophilic bacterium, was isolated from highly saline petroleum-contaminated soil in Shandong province, China 12 . It is highly effective in degrading many PAHs, such as naphthalene, anthracene, and phenanthrene, under broad salinities (0.1-15%) and varying pH (6.0-10.0) 13 . Salicylic acid was also utilized by Martelella sp. strain AD-3. At the same time, significantly, high activity of GDO was observed 13 . Furthermore, genes related to degrading gentisic acid has been annotated in the genome sequence of strain AD-3 14 .
In this study, we cloned, expressed, and characterized the gene encoding gentisate 1,2-dioxygenase from Martelella AD-3. Moreover, through site-directed mutagenesis, we determined that four His residues in GDO from strain AD-3 are important for its enzymatic activity. Finally, the source of the two oxygen atoms added to the maleylpyruvate was also analyzed.

Results
Gene cloning, identification, and amino acid sequence analysis. The 1,077-bp gentisate 1,2-dioxygenase gene gdo, was found by mining the genome sequence of strain AD-3 14 . Amino acid sequence alignment of GDO from strain AD-3 with other related proteins was performed with the Vector NTI program. Compared with other known sequences, the highest identity (70.8%) was found with Rhodococcus opacus CIR2 (RnoH, AB186916) 3 , and 60.4% identity with Corynebacterium glutamicum ATCC 13032 2 . Amino acid sequence of the AD-3 shared 30.9% and 29.1% sequence identities with two amino acid sequences from Pseudomonas alcaligenes NCIB 9867 7 . Compared with other two Haloferax strains (Haloferax sp. D1 and Haloferax sp. D1227) 8 , strain AD-3 had 24.9% and 24.2% identity, respectively. A phylogenetic tree was constructed with GDO proteins from 15 other strains and demonstrated that the protein from strain AD-3 is most closely related to Rhodococcus opacus R7 3 and Rhodococcus jostii RHA1 15 (Fig. 1B).   Fig. 2A). Protein GDO was purified, and obtained at 12 mg from 1 L LB medium. SDS-PAGE showed the purified protein to be approximately 38 kDa in size ( Fig. 2A). Analysis of the eluted fraction by Native-PAGE produced a prominent band of ~120 kDa (Fig. 2B). When stored at − 20 °C for 98 h, enzymatic activity was lost.
Enzyme kinetics of GDO. First, full wavelength scanning of GDO was performed. GDO showed a characteristic absorption peak at 279 nm, which is not the characteristic absorption peak FAD or NADH. Secondly, the result of enzyme kinetics showed that the absorbance at 330 nm steadily increased with time in 1 s, without any transition (Fig. 4). Finally, in order to determine the K m and k cat values, GDO activity was assayed at various gentisate concentrations (0-500 mM). The K m value for gentisate was determined to be 26.64 μ M and the k cat value was 161.29 s −1 .
Conserved His residue mutations. Multiple amino acid sequence alignment of GDO sequences from various strains showed the presence of four highly conserved His residues at positions 155, 157, 167, and 169. We designed primers to generate mutants with His to Ala amino acids changes. The resultant mutant plasmids (pET28a-H155A, pET28a-H157A, pET28a-H167A, and pET28a-H169A) were expressed in E. coli. The mutant proteins were then purified and the enzyme activity was assayed under the same conditions as for wild type GDO. No enzymatic activity was observed for the four mutant proteins compared to the wild type (Fig. S1).    increased about 6.5 times in the process of degrading phenanthrene and 80.4 times in response to gentisic acid 16 . (Fig. S2).

Discussion
In recent years, the issue of PAHs pollution has initiated concern among the general public. Specifically, PAHs pollution at high concentrations, in high salinity conditions, and combined pollution 17 has been of significant concern. Finding microorganisms with the ability to degrade PAHs in high salinity conditions is very important, however, in general, it is difficult for microorganisms to survive under these conditions. A PAH-degrading bacterial strain was previously isolated from highly saline petroleum-contaminated soil 13 and named as halophilic Martelella AD-3. Previous studies indicated that strain AD-3 has a wide spectrum for PAHs substrate degradation though gentisate pathway and a broad range of salinity from 0.1-15% 14 . Therefore, dissecting the specific features of GDO from AD-3 may be helpful to understand the enzymatic mechanisms of GDO.
In this study, gentisate 1,2-dioxygenase from strain AD-3 was purified and characterized. The maximum enzyme activity was observed at 12% salinity. Optimal activity of GDO from Haloferax sp. D1227 was reported at 2 M KCl or NaCl (10.4% salinity) 9 . Halophilic proteins function well in high-salt conditions, possessing additional acidic residues (glutamic acid and aspartic acid) 18 . Compared to the amino acid sequence of GDO from Haloferax sp. D1227, the sequence from AD-3 contains 16.7% additional acidic residues. This may explain why the higher salinity of 12% was the best condition for its enzymatic activity.
A previous study reported that Fe 2+ increased GDO activity by approximately 160% at Fe 2+ concentrations between 0.05-0.10 mM, for Pseudomonas alcaligenes NCIMB 9867 10 . At similar concentrations of Fe 2+ , the activity of GDO from E. coli O157:H7 was increased by 115% 21 . For the GDO from strain AD-3, the addition of Fe 2+ to a final concentration of 0.25 mM only increased enzymatic activity by 34%. The addition of Fe 2+ was also able to restore part activity of the comparely lost activity GDO from strain AD-3. Cu 2+ , Mn 2+ can activate GDO from strain AD-3, however the addition of 1 mM Cu 2+ , or 10 mM Mn 2+ can completely inactivate the GDO from K. pneumoniae 21 . The enzyme from Pseudomonas alcaligenes NCIB 9867 was inactivated by 5 mM Cu 2+7 . The crystal structure of E. coli-encoded GDO demonstrates the occupation of three of the possible six iron coordination sites by protein residues, His104, His106, and His145 21 . In this study, in order to assess whether the effect of Fe 2+ on GDO from strain AD-3 is similar to that of others, we performed site-directed mutagenesis, changing each of the four highly conserved His residues to Ala residues. The results suggest that all four highly conserved His residues are crucial for GDO activity. However, in Pseudomonas alcaligenes NCIMB 9867 22 , Silicibacter (Ruegeria) pomeroyi DSS-3 (AAV97252.1) 11 , Klebsiella pneumoniae M5a1, and Ralstonia sp. strain U2 1 , site-directed mutagenesis was performed, changing His residues to Asp residues, based on the results of error-prone PCR mutagenesis of gdo from Pseudomonas alcaligenes NCIB 9867 6,23 . It is possible that GDO could lose its activity when any one of the four His residues was replaced by Asp residues. His and Asp can carry different charges, so the results of the mutagenesis study are not completely convincing. The results in this present study provide further convincing evidence to demonstrate the importance of the four His residues by changing the four His residues to Ala residues.
In this study, we confirmed the source of the two oxygen atoms introduced into maleylpyruvate by 18 O isotope experiments and subsequent LC-MS analysis. Results suggested that the two oxygen atoms are derived from H 2 O and O 2 . According to the result, we speculated acquiring the oxygen from the O 2 is 'easier' than acquiring it from H 2 O. Because of the traces of 16 O 2 , the one 18 O labeled maleylpyruvate m/z 187.0126 was observed (Fig. 5D) In summary, we cloned, sequenced, and characterized a novel gene gdo in the degradation of PAHs from Martelella strain AD-3. These findings will expand our knowledge of the mechanisms of gentisate 1,2-dioxygenase enzymatic activity.

Materials and Methods
Chemicals and media. Gentisic acid (98% purity) was purchased from Sangon Biotech (Shanghai, China). All other reagents and solvents used were analytical grade and the highest purity available. Luria-Bertani (LB) broth (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) was used for both culturing and cloning. Solid agar plates were prepared with the addition of 1.5% (w/v) agar to the liquid medium. 18  Bacterial strains, plasmids, and growth conditions. Escherichia coli DH5α was used as host for recombinant plasmids, and E. coli BL21(DE3) (Invitrogen, Carlsbad, CA, USA) was used for expression. The gene gdo was amplified from the halophilic Martelella strain AD-3. Gene expression plasmids pET28a were obtained from Invitrogen (Invitrogen, Carlsbad, CA, USA). All recombinant cells were grown in LB broth or LB agar plates (15% [w/v]) containing 50 mg/L kanamycin 8 .
Expression of His 6 -tagged GDO. The gene gdo was PCR amplified from the genomic DNA of strain AD-3 with Prime STAR HS DNA polymerase (Takara co. Ltd., Dalia, China) 16 . Primers were designed such that the forward primer contains an NdeI site and the reverse primer contains a HindIII site. The primer sequences were as follows, forward, 5′ -GCCGCATATGAACATGATGATGCCTGAAGA-3′ and reverse, 5′ -ATTAAGCTTTCATGCGTCTGCGTCTTCGACC-3′ (NdeI and HindIII recognition sites are underlined). PCR amplification was performed with 100-μ l reaction mixtures containing 50 pmol of each primer, 10 μ l of a deoxynucleoside triphosphate mixture, 250 ng of template DNA, and 50 μ l of 2 × Prime Star buffer. PCR was carried out by using the following program, 5 min at 94 °C and then 30 cycles of 30 s at 94 °C, 30 s at 58 °C, and 2 min 30 s at 72 °C. The PCR products were purified, digested with NdeI and HindIII, and then ligated into pET28a (Novagen Corp., Germany), which had been double digested with the same restriction enzymes. The pET28a-gdo plasmid was transformed into E. coli BL21(DE3). DNA restriction enzymes and T4 DNA ligase were purchased from New England Biolabs 26 . E. coli BL21(DE3) cells containing pET28a-gdo, were cultured at 37 °C in LB medium containing 50 μ g/ml kanamycin to an OD 600 of 0.6-0.8. Then, after adding IPTG to a final concentration of 1 mM, the cultures incubated for 8 hours at 16 °C or 20 °C or 30 °C to express GDO. The cells were harvested and resuspended in binding buffer (25 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0) at an OD 600 of 30. Cells were then lysed by sonication on ice, and centrifuged at 10,000 × g for 20 min to separate the soluble cell lysate from the insoluble membrane and protein aggregates 5 .
Purification of GDO. The supernatant was filtered through a 0.22 μ m filter and the resultant filtrate was applied to a 5-ml column of Ni-NTA agarose (GE, Healthcare, Little Chalfont, UK), which had been equilibrated with the binding buffer. After a wash with 30 ml of washing buffer (25 mM Tris-HCl, 300 mM NaCl, 70 mM imidazole, pH 8.0), His 6 -tagged GDO was eluted from the column with elution buffer (25 mM Tris-HCl, 300 mM NaCl, 200 mM imidazole, pH 8.0). All purification steps were carried out at 4 °C.
Enzyme assays and protein determination. Gentisate 1,2-dioxygenase activity was spectrophotometrically assayed at 330 nm by measuring maleylpyruvate formation 5 . Activity was assayed in 1 ml of reaction mixture containing 0.46 mM gentisate in 0.1 M phosphate buffer, pH 7.4, at 23 °C with a UV-2550 spectrophotometer (Shimadzu, Kyoto, Japan). A molar extinction coefficient of 10.8 × 10 3 M/cm was used to calculate specific activity 20 . One enzyme unit was defined as the amount of enzyme that produces 1 mmol of maleylpyruvate per min at 23 °C 7 . Enzyme activity was assayed 5 min after adding the enzyme to the reaction mixture.
Rapid reaction kinetics. The enzyme kinetics was measured with an SFM 4000 stopped flow apparatus (BioLogic, France). Constant temperature was maintained in a JULABO model F21 temperature-controlled bath (Julabo, Seelbach, Germany). Spectral scans were recorded with a TIDAS S 300 K diode array detector (J&M Analytik AG, Germany) 27 . Experimental parameters were set as follows, enzyme/substrate solutions, 1/1; UV absorbance spectra, 330 nm; injection rate, 20 μ l/s; detection times, 10 ms to 10 s; temperature, 4 °C. Additionally, the enzyme concentration was no lower than 5 mg/ml and the gentisic acid were the same as in the enzyme assays.
Site-directed mutagenesis. Site-directed mutagenesis was performed using a recombinant PCR method. Primers are listed in Table 1. Mutant genes were subcloned into a pET28a vector between the NdeI and HindIII restriction sites, separately. All mutant strains were analyzed by sequencing to confirm disruption of the target gene 28 .
Scientific RepoRts | 5:14307 | DOi: 10.1038/srep14307 DNA and amino acid sequence analysis. Amino acid sequences of GDO from other strains were obtained from GenBank. All homology searches were carried out on the NCBI BLAST server (http,// www.ncbi.nlm.nih.gov/BLAST) with the nucleotide BLAST and protein BLAST. These obtained GDO sequences were then compared with the sequence from AD-3. Conserved binding domain searches were performed using Vector NTI DNA analytical software (version 11.0).

O isotope experiments.
To ascertain the origin of the two oxygen atoms added onto maleylpyruvate, H 2 18 O was added to the solvent for the gentisate reaction or 18 O 2 was added to the anaerobic environment. Then, the assay mixtures of dried GDO powder and gentisic acid dissolved in H 2 18 O were incubated at 30 °C for 20 min. Another assay mixture dissolved in H 2 16 O was incubated at the same temperature for 20 min, injected with 18 O 2 at the beginning. As a control, another mixture dissolved in H 2 16 O was prepared for reacting in the 16 O 2 atmosphere. After termination of the reaction by adding 2 volumes of ethanol, samples were analyzed using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) (samples were prepared by filtration) 28 , the negative mode of ESI-MS analysis was used to monitor the products.
Liquid chromatography-mass spectrometry (LC-MS) analysis. Identification of GDO degradation products was carried out by LC-MS (Agilent, Pale Alto, CA, USA) using ESI in the negative ion mode. Substrate solutions were prepared by adding two volumes of ethanol to precipitate the enzyme and filtered through a 0.22-μ m Millipore filter 80 min after adding 0 μ l or 1 μ l GDO enzyme. Then, the samples were automatically injected (5 μ l) into the high performance liquid chromatography system. Separation was achieved by ion-pair chromatography on a Luna C18 5 μ m column ( RT-qPCR analysis. Total RNA was isolated from strain AD-3, which was cultured in glycerol medium or induced medium with the presence of phenanthrene or gentisic acid, using an RNeasy mini kit (TIANGEN, China) 16 . Reverse transcription PCR (RT-PCR) was performed using a Prime Script one-step RT-PCR kit (Takara, Japan). Quantitative PCR reactions were carried out using 0.2 ml qPCR tubes (Bio-Rad) and a Chromo 4 real-time PCR thermocycler (Bio-Rad). Reaction was performed by using 20-μ l reaction mixtures containing 9 μ l PerfeCTa SYBR Green Fast Mix (TIANGEN, China), 0.4 μ l of each primer, 10 ng DNA sample and DNase free water to a final volume of 20 μ l. The standard curve for each pair of primer was constructed with a tenfold dilution of genomic DNA from strain AD-3 26 . The 16S rRNA gene was used as control. For gene gdo, the thermocycler program used for qPCR was as follow: 95 °C for 3 min, 34 cycles of 20 s at 95 °C, 10 s at 63.2 °C/50.9 °C, and 15 s at 68 °C, then 95.0 °C for 1 min, 55.0 °C 1 min and 5 s, stored at 95 min. The primers for RT-qPCR were 5′ -AAGAGGTAAGTGGAATTG-3′ and 5′ -CAGTAATGGACCAGTAAG-3′ for 16S rRNA, 5′ -ATGATGATGCCTGAAGACA-3′ and 5′ -GAGCGGATTGAGGTGATT-3′ for the gene gdo. The threshold cycle (Ct) values for gene gdo from three different conditions, were normalized to the reference gene, 16S rRNA gene. The relative expression level was calculated by using the 2 -ΔΔCt method, where Δ Δ Ct = (Ct target-Ct 16S rRNA) INDUCTION -(Ct target-Ct 16S rRNA) CONTROL 16 .