Characterization of a Novel Nicotine Hydroxylase from Pseudomonas sp. ZZ-5 That Catalyzes the Conversion of 6-Hydroxy-3-Succinoylpyridine into 2,5-Dihydroxypyridine

Abstract

A novel nicotine hydroxylase was isolated from Pseudomonas sp. ZZ-5 (HSPH_ZZ). The sequence encoding the enzyme was 1206 nucleotides long, and encoded a protein of 401 amino acids. Recombinant HSPH_ZZ was functionally overexpressed in Escherichia coli BL21-Codon Plus (DE3)-RIL cells and purified to homogeneity after Ni-NTA affinity chromatography. Liquid chromatography-mass spectrometry (LC-MS) analyses indicated that the enzyme could efficiently catalyze the conversion of 6-hydroxy-3-succinoylpyridine (HSP) into 2,5-dihydroxypyridine (2,5-DHP) and succinic acid in the presence of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). The kinetic constants (K_m, k_cat, and k_cat/K_m) of HSPH_ZZ toward HSP were 0.18 mM, 2.1 s⁻¹, and 11.7 s⁻¹ mM⁻¹, respectively. The optimum temperature, pH, and optimum concentrations of substrate and enzyme for 2,5-DHP production were 30 °C, 8.5, 1.0 mM, and 1.0 μM, respectively. Under optimum conditions, 85.3 mg/L 2,5-DHP was produced in 40 min with a conversion of 74.9%. These results demonstrated that HSPH_ZZ could be used for the enzymatic production of 2,5-DHP in biotechnology applications.

1. Introduction

Nicotine is the principal alkaloid in tobacco plants, and is responsible for smoking addiction and several diseases such as cancer and pulmonary disease [1,2,3]. The manufacturing of tobacco products and all activities involving tobacco produce large amounts of solid or liquid waste with a high nicotine content. Therefore, nicotine is the primary toxic substance in tobacco wastes [3,4]. These wastes have been designated as “toxic and hazardous wastes” under European Union Regulations [5]. Since tobacco waste is a major problem for public health and the environment, it is important to establish appropriate detoxification methods. Microbial degradation of nicotine provides a method to treat such wastes [6,7]. Several microorganisms have been found to degrade nicotine, including Arthrobacter, Pseudomonas, Agrobacterium tumefaciens S33, Aspergillus oryzae, Sphingomonas sp., Acinetobacter sp., Shinella sp. strain HZN7, Rhodococcus sp., and Cellulomonas sp. [3,8,9,10,11,12,13,14].

Recent studies have reported methods to synthesize chemicals of pharmaceutical importance from nicotine, such as (S)-macrostomine, nicotinonitrile, and fused-ring nicotine derivatives [15,16,17,18]. Dihydropyridines are important precursors in medicinal chemistry, and have been used to produce compounds with antitumor activity for cancer therapy, antidiabetic agents to treat vascular disorders, and HIV protease inhibitors [19,20,21,22]. However, the lack of efficient methods to synthesize 2,5-dihydropyridine has restricted studies on its biological activity and applications [18]. The traditional chemical method to produce 2,5-dihydropyridine (2,5-DHP) is complex, creates hazardous byproducts, and has potentially undesirable side reactions and a high energy cost. Therefore, the enzymatic synthesis of 2,5-dihydropyridine represents an attractive alternative to chemical synthesis, because of its high specificity, high efficiency, and low production of pollutants.

In this study, a novel HSP (6-hydroxy-3-succinoylpyridine) hydroxylase was purified from Pseudomonas sp. ZZ-5 (HSPH_ZZ), and then its encoding gene was cloned and expressed in bacterial cells. The recombinant HSPH_ZZ exhibited high activity to convert HSP to 2,5-DHP. The optimum concentrations of substrate and enzyme for the formation of 2,5-DHP were determined, and the kinetic parameters and catalytic properties of the recombinant HSPH_ZZ were characterized.

2. Results and Discussion

2.1. Purification and Identification of HSPH_ZZ

The HSPH_ZZ with HSP hydroxylase activity was purified to homogeneity from cells of Pseudomonas sp. ZZ-5 using the steps shown in

Table 1. Purification of a novel HSP (6-hydroxy-3-succinoylpyridine) hydroxylase from Pseudomonas sp. ZZ-5 (HSPH_ZZ).

Step	Total Protein (mg)	Total Activity (U)	Specific Activity (U/mg)	Fold	Yield (%)
Crude cell extract	672.6	195.4	0.29	1	100
(NH4)2SO4 precipitation	542.3	173.5	0.31	1.1	88.8
DEAE sepharose Phenyl sepharose	75.2 22.3	100.3 54.5	1.3 2.4	4.6 8.4	51.3 27.8
Superdex-200	5.1	26.1	5.1	17.6	13.4

Values given are the average of three replications.

. The enzyme was purified 17.6-fold to give a yield of 13.4% and a specific activity of 5.1 U/mg. The molecular mass of the purified enzyme was estimated to be 45 kDa by SDS-PAGE (Figure 1), similar to that of HSP hydroxylases from Pseudomonas putida S16 and Agrobacterium tumefaciens S33 [23,24].

To identify the reaction catalyzed by the purified enzyme, the products of the HSPH_ZZ oxidation reaction were analyzed by liquid chromatography-mass spectrometry (LC-MS). The analyses of the reaction products revealed m/z values of 110.0, 117.1, and 194.0, which corresponded to the calculated molecular mass of 2,5-DHP (C₅H₅NO₂, 111.10), succinic acid (C₄H₆O₄, 118.09), and HSP (C₉H₉NO₄, 195.17), respectively (Supplementary Figure S1). These results demonstrated that HSPH_ZZ has high HSP hydroxylase activity to convert HSP to 2,5-DHP and succinic acid (Figure 2). To our knowledge, HSPH_ZZ is only the second example of an HSP hydroxylase from Pseudomonas to-date.

2.2. Gene Cloning and Sequence Analysis of HSPH_ZZ

The N-terminal amino acid sequence of purified HSPH_ZZ was determined to be M-S-G-H-L-R-V-I-I-I-V-G-G-G-P by the automated Edman degradation technique. The DNA fragment encoding HSPH_ZZ was directly amplified by PCR using the genomic DNA of Pseudomonas sp. ZZ-5 as the template. The HSPH_ZZ sequence (1206 bp) was found to encode a 44.3 kDa protein composed of 401 amino acids. The amino acid sequence of HSPH_ZZ showed high identity with HSPHs from P. putida S16 (Accession No. WP_013973865.1, 86% identity), Pseudomonas sp. JY-Q (Accession No. WP_064613869.1, 85% identity), Pseudomonas sp. EGD-AK9 (Accession No. WP_031302470.1, 83% identity), and A. tumefaciens (Accession No. WP_031302470.1, 60% identity), respectively. Multiple sequence alignment analyses revealed that HSPH_ZZ contained the conserved motif Gly-Ala-Glu-Gly-Ala (amino acids 166–170), which is associated with FAD- and NAD(P)H-binding [25] (Supplementary Figure S2). At the amino acid sequence level, HSPH_ZZ also showed high identity to p-nitrophenol monooxygenases from Acidovorax sp. NO-1 (58%), Sphingomonas melonis TY (52%), and Massilia putida (43%). However, HSPH_ZZ did not show any detectable activity with p-nitrophenol as the substrate (data not shown).

2.3. Expression of Recombinant HSPH_ZZ in Escherichia coli BL21-Codon Plus (DE3)-RIL

The recombinant plasmid pET22b–HSPH_ZZ was constructed to determine the catalytic properties of recombinant HSPH_ZZ. The recombinant HSPH_ZZ was expressed in E. coli BL21-Codon Plus (DE3)-RIL cells and purified to homogeneity after Ni–NTA affinity and Superdex 200 gel filtration chromatography. The purified recombinant HSPH_ZZ with a polyhistidine (6× His) tag at the C terminus was visible as a major band with a calculated mass of 45 kDa in SDS-PAGE analyses (Figure 1). The specific activity of the purified recombinant HSPH_ZZ was 31.8 U/mg—6.2-fold that of the HSPH isolated from the original strain Pseudomonas sp. ZZ-5 (Supplementary Table S1).

2.4. Effect of Temperature and pH on Activity of the Recombinant HSPH_ZZ

The effects of pH (5.5–10) and temperature (5–45 °C) on the hydroxylase activity of the recombinant HSPH_ZZ were determined using the standard enzyme assay. The optimum temperature and pH of HSPH_ZZ were determined to be 30 °C and 8.5, respectively (Figure 3). The pH optimum was higher than that of reported HSP hydroxylases from P. putida S16 and A. tumefaciens S33 (pH 8.0) [23,24]. The thermostability of HSPH_ZZ was evaluated at three different temperatures (30 °C, 35 °C, and 40 °C) with increasing incubation times up to 120 min. Most of the enzyme activity was maintained after incubation at 30 °C for at least 120 min, whereas incubation at 40 °C for 30 min reduced to approximately 40% of the maximum activity. The pH stability of HSPH_ZZ was determined at pHs ranging from 5.5 to 10.0, and the results demonstrated that over 50% of its maximal activity was maintained from pH 7.0 to 9.0 (Supplementary Figure S3).

2.5. Effect of Enzyme and Substrate Concentration on 2,5-DHP Production

As shown Figure 4, the effects of enzyme (0.2–2.0 μM) and substrate concentration (0.25–2.0 mM) on 2,5-DHP production were investigated. In a reaction system containing 1.0 mM HSP, the production of 2,5-DHP increased with increasing enzyme concentration from 0.2 to 1.0 μM, and then reached a plateau at about 1.0 μM. Thus, 1.0 μM was considered to be the optimum enzyme concentration for 2,5-DHP production (Figure 4). The presence of excess enzyme could result in enzyme agglomeration and diffusion problems, which could decrease the reaction efficiency [26]. The maximum production of 2,5-DHP was observed at 1 mM HSP (Figure 4). Any further increase or decrease in the HSP concentration resulted in a decrease in 2,5-DHP production. Thus, increasing the HSP concentration (from 0.25 to 1 mM) facilitated contact between the enzyme and the substrate in the reaction system [27]. The Michaelis–Menten equation was applied for catalytic kinetic analysis. The kinetic parameters of HSPH_ZZ for HSP were calculated under optimal conditions (at 30 °C and pH 8.5 in 20 mM Tris-HCl buffer) and a nicotinamide adenine dinucleotide (NADH) concentration of 1.0 mM. The K_m, k_cat, and k_cat/K_m of HSPH_ZZ for HSP were 0.18 mM, 2.1 s⁻¹, and 11.7 s⁻¹ mM⁻¹, respectively (

Table 2. Kinetic parameters of HSPH_ZZ for the substrate of HSP or NADH ^a.

Substrate	kcat(s−1)	Km(mM)	kcat/Km(s−1 mM−1)
HSP	2.1 ± 0.3	0.18 ± 0.04	11.7 ± 1.5
NADH	1.3 ± 0.2	0.23 ± 0.03	5.7 ± 1.1

^a The values are means of three independent experiments.

). When the HSP concentration was 1.0 mM, the K_m, k_cat, and k_cat/K_m of HSPH_ZZ for NADH were 0.23 mM, 1.3 s⁻¹, and 5.7 s⁻¹ mM⁻¹, respectively (Table 2).

2.6. Effect of Metal Ions, Organic Solvents, and Detergents on Enzymatic Activity

The effect various metal ions (Mg²⁺, Zn²⁺, Cu²⁺, Ca²⁺, Mn²⁺, Ni²⁺, Co²⁺, Fe²⁺, Na⁺, and K⁺) at 5 mM on enzymatic activity was investigated by the standard enzyme assay (

Table 3. Effect of various metals on the enzymatic activity of HSPH_ZZ ^a.

Metals or Inhibitors	Concentration	Relative Activity (%)
None	-	100
Mg2+	5 mM	86 ± 3
Zn2+	5 mM	27 ± 2
Cu2+	5 mM	23 ± 1
Ca2+	5 mM	92 ± 2
Mn2+	5 mM	87 ± 4
Fe2+	5 mM	13 ± 2
Ni2+	5 mM	81 ± 3
Co2+	5 mM	31 ± 3
K+	5 mM	101 ± 5
Na+	5 mM	98 ± 2
EDTA	0.5 mM 5 mM	79 ± 2 85 ± 2

^a The values are means of three independent experiments.

). The activity of HSPH_ZZ was not significantly affected by Mg²⁺, Ni²⁺, Ca²⁺, Mn²⁺, K⁺, Na⁺, or the chelator EDTA (0.5 mM and 5 mM), indicating that this enzyme was not a metalloprotein. However, Cu²⁺, Co²⁺, Zn²⁺, and Fe²⁺ significantly inhibited enzyme activity. As shown in

Table 4. Effect of various organic solvent and detergents on the activity of HSPH_ZZ ^a.

Metals or Inhibitors	Concentration (%)	Relative Activity (%)
None	—	100
Methanol	20 (v/v)	61 ± 2
Acetone	20 (v/v)	59 ± 4
Chloroform	20 (v/v)	33 ± 2
Formaldehyde	20 (v/v)	23 ± 3
Toluene	20 (v/v)	28 ± 5
DMF	20 (v/v)	65 ± 2
DMSO	20 (v/v)	93 ± 5
Tween 20	1 (w/v)	86 ± 5
Tween 40	1 (w/v)	110 ± 4
Tween 80	1 (w/v)	91 ± 1
Triton X-100	1 (w/v)	48 ± 3
Span 20	1 (w/v)	12 ± 4
Span 40	1 (w/v)	17 ± 5

^a The values are means of three independent experiments.

, the effects of methanol, acetone, formaldehyde, Dimethylformamide (DMF), chloroform, and toluene at 20% (v/v) also markedly inhibited enzyme activity, while more than 90% of HSPH_ZZ activity was retained after incubation with DMSO. Incubation with Triton X-100, Span 20, and Span 40 at 1% (w/v) reduced the hydroxylase activity by less than 50% (Table 4). The enzyme activity was not reduced by adding Tween 20, Tween 40, or Tween 80 at 1% (w/v).

2.7. 2,5-DHP Production from HSP by HSPH_ZZ under Optimum Conditions

The production of 2,5-DHP from HSP by HSPH_ZZ was investigated under optimum conditions (at 30 °C and pH 8.5 in 20 mM Tris-HCl buffer), with HSPH_ZZ at a concentration of 1 μM in the standard enzyme assay. The enzyme produced 2,5-DHP (85.3 mg/L) in 40 min with a conversion of 74.9% (w/w) (Figure 5). The 2,5-DHP decomposed over time in the reaction solution, indicating that it was unstable and probably sensitive to oxidation. The amount of HSP consumed in the enzyme reaction in 40 min was 161 mg/L (0.83 mM) out of the initial 200 mg/L. Based on the conversion stoichiometry, the estimated amount of 2,5-DHP produced was 92.1 mg/L, corresponding to a conversion yield of 80.9%. Thus, the amount of 2,5-DHP lost during the reaction was 6.8 mg/L (amount produced minus amount detected). The rate of HSP catalysis by HSPH_ZZ was higher than that of the HSP hydroxylase from A. tumefaciens S33 (69.7% at 35 °C and pH 8.0 in 50 min) [28]. These results demonstrated that HSPH_ZZ has strong activity to produce 2,5-DHP HSP, making it a potential candidate for enzymatic transformation of HSP into 2,5-DHP in commercial applications.

3. Experimental Section

3.1. Chemicals, Strains, and Plasmids

Using nicotine as the sole source of carbon and nitrogen, Pseudomonas sp. ZZ-5 was isolated from soil samples obtained from a field under continuous tobacco cropping in Henan, P.R. China. The strain was cultured in nicotine medium containing (per liter) 22.8 g K₂HPO₄·3H₂O, 6.8 g KH₂PO₄, 0.5 g MgSO₄·7H₂O, and 1 mL trace elements solution. The trace elements solution contained 0.05 g CaCl₂·2H₂O, 0.05 g CuCl₂·2H₂O, 0.008 g MnSO₄·H₂O, 0.004 g FeSO₄·7H₂O, 0.1 g ZnSO₄, 0.1 g Na₂MoO₄·2H₂O, and 0.05 g Na₂WO₄·2H₂O [9,29]. The plasmid pET22b and E. coli strain BL21-CodonPlus (DE3)-RIL were respectively bought from Novagen (Madison, WI, USA) and Stratagene (La Jolla, CA, USA). Pyrobest DNA polymerase, restriction enzymes, and the DNA ligation kit were purchased from Takara Biotechnology (Dalian, China). The DEAE-anion exchange column, phenyl-sepharose column, nickel columns, and Superdex 200 gel filtration columns were from GE Healthcare (Buckinghamshire, UK), and 2,5-DHP was purchased from SynChem OHG (Altenburg, Germany). Chromatographic-grade succinic acid was purchased from J&K Scientific Ltd. (Beijing, China). We obtained HSP from Professor Hongzhi Tang at Shanghai Jiao Tong University. All other chemicals were of analytical grade and were obtained from Sangon (Shanghai, China).

3.2. Purification of HSPH_ZZ

Cells of Pseudomonas sp. ZZ-5 (approx. 15 g) were suspended in 50 mM Tris-HCl buffer (pH 8.0) and disrupted by sonication (40 kHz, 80–100 W). The supernatant was subjected to ammonium sulfate fractionation and yielded a precipitate at 50–70% saturation with (NH₄)₂SO₄. The mixture was allowed to stand overnight, and then the precipitate was collected by centrifugation at 20,000× g for 20 min. The precipitate was loaded onto a DEAE-anion exchange column (1 × 20 cm², GE Healthcare) equilibrated with 50 mM Tris-HCl buffer (pH 8.0). The column was washed with a linear salt gradient from 50 mM to 1 M NaCl. The fractions containing the target protein were collected at 300–400 mM and concentrated using a membrane with a 30-kDa MW cut off. The sample was applied to a phenyl-sepharose column (1 × 20 cm², GE Healthcare, Buckinghamshire, UK) equilibrated with 50 mM Tris-HCl buffer containing 1.0 M (NH₄)₂SO₄. The target protein was eluted at an (NH₄)₂SO₄ concentration of 0.8 M, desalted, and then enriched. Finally, the protein mixture was loaded onto a Superdex 200 gel filtration column (GE Healthcare, Buckinghamshire, UK), which was pre-equilibrated with 50 mM Tris-HCl (pH 8.0) containing 200 mM NaCl. The fraction size was 1 mL (flow rate, 1 mL/min). The peak fractions were determined by assaying enzymatic activity under standard conditions. The fractions with HSP hydroxylase activity were collected and analyzed by SDS-PAGE. The protein concentration was determined using the Bradford method.

3.3. Enzymatic Activity Assay

The activity of HSP hydroxylase was determined using the method of Tang et al. [23,30]. The standard reaction mixture, containing 10 mM FAD, 1 mM HSP, 0.5 mM NADH, and 20 mM Tris-HCl buffer (pH 8.5), was preincubated for 2 min and then the reaction was started by adding the purified enzyme. The enzymatic activity was measured at 30 °C for 30 min and the production of 2,5-DHP was monitored by liquid chromatography-mass spectrometry (LC-MS) analysis. The reaction products were characterized by an electron spray ionization (ESI) source on an AB Sciex Triple Quad 5500 mass spectrometer with an Agilent 1290 infinity LC system for UHPLC. The UHPLC analyses were performed with a ZORBAX Eclipse Plus-C18 column (column size, 150 × 4.6 mm; particle size, 5 μm; Agilent, Palo Alto, CA, USA) and a mobile phase of methanol:1 mM acetic acid (25:75, v/v; flow rate, 0.5 mL/min).

One unit of hydroxylase activity was defined as the amount of enzyme releasing 1 mol 2,5-DHP per min under standard conditions. Measurements were corrected for background hydrolysis in the absence of enzyme.

3.4. N-Terminal Amino Acid Sequence of HSPH_ZZ

After analysis by SDS-PAGE, the target protein was transferred onto a polyvinylidene difluoride membrane Hybond-P (Amersham Pharmacia Biotech, Orsay, France) and visualized with Coomassie brilliant blue staining. Then, the corresponding bands were excised, and the N-terminal and internal partial amino acid sequences of the protein were determined by the Sangon.

3.5. Gene Cloning and Construction of Expression Plasmid

The N-terminal amino acid sequence of HSPH_ZZ was used to search the GenBank database, and the sequences retrieved were used to design primers to amplify HSPH_ZZ. The gene encoding HSPH_ZZ was amplified using PCR with the following primers: forward, 5′-GCACATATGAGCGGACATCAGGATGTCATC-3′, and reverse, 5′-GCGGTCGACCAACTATGTCTGCATTAATTGCGG-3′ (NdeI and SalI sites underlined, respectively). Genomic DNA was extracted from Pseudomonas sp. ZZ-5 using a Gentra Puregene Yeast/bact. Kit B following the manufacturer’s instructions (Qiagen, Valencia, CA, USA) and used as the template for PCR amplification. The PCR conditions were as follows: 94 °C for 1 min, 60 °C for 1 min, 72 °C for 2 min, and a final extension at 72 °C for 10 min. The PCR product was subsequently purified, digested with NdeI and SalI, and then cloned into pET22b to generate pET15b–HSPH_ZZ. The DNA insert was sequenced to confirm that no unintended mutation had occurred.

3.6. Expression and Purification of Recombinant HSPH_ZZ

The plasmid pET15b–HSPH_ZZ was transformed into E. coli BL21-Codon Plus (DE3)-RIL cells for gene expression. The transformed cells were grown in LB broth containing 100 mg/L ampicillin and 34 mg/L chloramphenicol at 37 °C and with shaking at 150 rpm. When the OD₆₀₀ value of the cultures reached 0.4–0.6, isopropyl-β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM to induce gene expression. After further culture for 6 h at 30 °C, the cells were harvested by centrifugation, resuspended in buffer A (50 mM Tris-HCl, pH 7.8, 200 mM NaCl) and disrupted by sonication. Cell debris was removed by centrifugation at 20,000× g at 4 °C for 20 min. The soluble fraction was loaded onto a Ni²⁺-NTA agarose column (GE Healthcare, Buckinghamshire, UK) pre-equilibrated with buffer A. After washing with buffer containing 60 mM imidazole, the enzymes were eluted with buffer B (20 mM Tris-HCl, pH 7.8, 500 mM NaCl, 300 mM imidazole). The purified protein was analyzed by SDS-PAGE (12% polyacrylamide) and the protein concentration was determined using the Bradford method.

3.7. Catalytic Properties of Recombinant HSPH_ZZ

The optimum pH and temperature for recombinant HSPH_ZZ activity were determined using the standard enzymatic assay and LC-MS (AB Sciex, Framingham, MA, USA). The effect of pH on hydroxylase activity was examined at 30 °C within the pH range of 5.5 to 10.0. The effect of temperature (from 5 °C to 45 °C) on enzymatic activity was investigated at pH 8.5. The effects of several different metal ions, organic solvents, and detergents on the activity of HSPH_ZZ were determined by adding each metal salt (5 mM), organic solvent (20%, v/v), and detergent (1%, v/v) to the standard assay solution.

4. Conclusions

We have cloned a novel HSP hydroxylase from Pseudomonas sp. ZZ-5 (HSPH_ZZ), and expressed it in a bacterial cell system. The recombinant HSPH_ZZ showed strong enzymatic activity to convert HSP to 2,5-DHP in the presence of NADH and FAD. The effects of temperature, pH, and concentrations of substrate and enzyme for 2,5-DHP production were optimized. Under optimum conditions, HSPH_ZZ produced 85.3 mg/L 2,5-DHP from 200 mg/L of HSP in 40 min with a conversion rate of 74.9%. These results demonstrate that HSPH_ZZ could be a potential candidate for the enzymatic synthesis of 2,5-DHP in pharmaceutical applications.