Crystal structure of the γ-hydroxymuconic semialdehyde dehydrogenase from Pseudomonas sp. strainWBC-3, a key enzyme involved in para-Nitrophenol degradation
© Su et al.; licensee BioMed Central Ltd. 2013
Received: 6 May 2013
Accepted: 14 November 2013
Published: 19 November 2013
para-Nitrophenol (PNP) is a highly toxic compound with threats to mammalian health. The pnpE-encoded γ-hydroxymuconic semialdehyde dehydrogenase catalyzes the reduction of γ-hydroxymuconic semialdehyde to maleylacetate in Pseudomonas sp. strain WBC-3, playing a key role in the catabolism of PNP to Krebs cycle intermediates. However, the catalyzing mechanism by PnpE has not been well understood.
Here we report the crystal structures of the apo and NAD bound PnpE. In the PnpE-NAD complex structure, NAD is situated in a cleft of PnpE. The cofactor binding site is composed of two pockets. The adenosine and the first ribose group of NAD bind in one pocket and the nicotinamide ring in the other.
Six amino acids have interactions with the cofactor. They are C281, E247, Q210, W148, I146 and K172. Highly conserved residues C281 and E247 were identified to be critical for its catalytic activity. In addition, flexible docking studies of the enzyme-substrate system were performed to predict the interactions between PnpE and its substrate γ-hydroxymuconic semialdehyde. Amino acids that interact extensively with the substrate and stabilize the substrate in an orientation suitable for enzyme catalysis were identified. The importance of these residues for catalytic activity was confirmed by the relevant site-directed mutagenesis and their biochemical characterization.
para-Nitrophenol (PNP), which is widely used in the manufacture of medicines, pesticides, dyes, explosives, leather coloring, wood preservatives and rubber chemicals, is highly toxic to animal’s health. Through the respiratory system and skin it affects the blood, liver and central nervous system in the body. It can cause dizziness, rash, itching spam, anemia and various neurological symptoms . It contains a nitroso group in the structure that strongly attracts the electron of phenyl, thus PNP is difficult for degradation in the nature .Therefore, a number of studies have been triggered focusing on the microbial degradation of PNP [3–6] and two pathways have been clearly illustrated so far. One is the hydroquinone pathway that is usually found in Gram-negative bacteria. For example, in Moraxella sp, PNP monooxygenase converts PNP to hydroquinone via the potential intermediate p-benzoquinone [7, 8]. The other is the hydroxyquinol (1, 2, 4-trihydroxybenzene) pathway that is preferentially found in Gram-positive bacteria. For example, in Rhodococcus opacus SAO101, PNP is converted to hydroxyquinol via 4-nitrocatechol [9, 10].
Pseudomonas sp. strain WBC-3 utilizes either methyl parathion (O,O-dimethyl O-p-nitrophenol phosphorothioate) or PNP as the sole source of carbon, nitrogen and energy for survival . This bacterium degrades PNP through the hydroquinone pathway. First, PNP is converted to p-benzoquinone by PNP 4-monooxygenase (PnpA) and p-benzoquinoneis further reduced to hydroquinone by p-benzoquinone reductase (PnpB). Next, hydroquinone dioxygenase (PnpCD) converts hydroquinone to γ-hydroxymuconic semialdehyde, which is then converted to maleyacetate by the NAD dependent γ-hydroxymuconic semialdehyde dehydrogenase (PnpE). Finally, maleylacetate reductase (PnpF) catalyzes the conversion of maleyacetate to β-ketoadipate before entering the TCA cycle.
In contrast to the reasonably thorough biochemical and genetic characterization of the PNP degradation, the structural mechanism of each reaction step in the pathway remains unclear. In order to understand the structural basis required for the activity of the enzyme, we embarked on structure determination of these enzymes that catalyze the PNP degradation process.
Recently, the primary X-ray analysis of PnpA in P.putida DLL-E4 was reported. In which PnpA was crystallized and the diffraction data was obtained . However, there was no further structural report about the enzyme or other molecules involved in the hydroquinone pathway. PnpE belongs to NAD dependent aldehyde dehydrogenase (ALDH) super-family which can catalyze the aldehydes oxidation to corresponding carboxylic acids [13–15]. To date, some crystal structures of NAD (P) + - dependent ALDH family have been reported [16–20]. These enzymes exhibit wide differences in substrate and cofactor specificity. Sequence alignments of them with PnpE gave amino acid sequence identities ranging from 23% to 45%. Among them mitochondrial aldehyde dehydrogenase from bovine gives the highest sequence identity with PnpE.
This super family involves highly conserved residues Cys which was essential nucleophile and Glu that was the general base necessary to activate Cys for the dehydrogenase reaction. The substrate of PnpE is γ-hydroxymuconic semialdehyde which is unstable in air. So far the catalytic mechanism of this substrate has not been reported. In the current study, we determined the crystal structure of Pseudomonas sp. strain WBC-3 apo-PnpE at 2.7 Å resolution and its complex with NAD at 3.1 Å resolution. Through enzyme-substrate docking-guided point mutations, we identified the active site of PnpE and studied its catalytic mechanism.
Bacterial strains, chemicals, media and culture conditions
Pseudomonas sp. Strain WBC-3 genomic DNA obtained from Wu Han Institute of Virology, Chinese Academy of Science. Escherichia coli strain BL21(DE3) used as expression host, which was cultured at 37°C in lysogeny broth (LB) and transformed as described by Sambrooket al. . All chemical products used in the experiment were purchased from Sigma Chemical Co. (St Louis, MO, USA).
Gene cloning and oligonucleotide-directed mutagenesis sequencing
pnpE gene from Pseudomonas sp. strain WBC-3 was cloned into NdeI and XhoI sites of pET29b (Novagen). Nine PnpE mutants (C281A, E247K, F150A, W157A, H275E, F447A, N149A, F154A, I282D) were produced using the two-step PCR strategy  and were confirmed by DNA sequencing.
Protein expression and purification
The E.coli cells were cultured in the LB medium containing 100 μg/mL ampicillin until OD600 reached 0.8 and were then induced with 1 mM IPTG overnight at 15°C. The cells were harvested by centrifugation. Cells lysis was achieved by sonication method. After centrifugation at 28,000 × g for 45 min, the supernatant was applied to Ni-NTA column. The His-tagged PnpE was eluted with elution buffer (10 mM Tris–HCl pH 8.0, 100 mM NaCl, and 250mM imidazole). The purification process was then followed by anion exchange on a Source-Q column and finally applied to size-exclusion chromatography with Superdex-200 column.
Enzyme activity assays
The catalytic activity of PnpE was measured by cascade reactions. The biosynthesis of γ-hydroxymuconic was performed in a total volume of 100 μL system containing 0.1 mM hydroquinone, 5.6μg hydroquinone dioxygenase PnpCD (PnpCD was expressed and purified in the same way as described in section 2.3) and 0.1 mM FeSO4 and 100mM Tris–HCl (pH 8.0) at 25°C. The absorbance of the reaction mixture was monitored at 290 nm until no further decrease was observed (about 10 min). At this point, the product solution can be used as the substrate for the next step to test PnpE activity. The assay of PnpE was performed immediately after the production of γ-hydroxymuconic in 100μL reaction system containing 5.2 μg purified PnpE, 10 μL γ-hydroxymuconic (produced by biosynthesis method), 0.1 mM NAD and 100 mM Tris–HCl (pH 8.0). The reaction was incubated at 25°C for 15 min. PnpE activity was determined by measuring the absorbance increase at 340 nm due to NADH formation.
Crystallization and data collection
Protein concentration was adjusted to 10 mg/mL before setting up crystallization screens at 20°C. Initially the native crystals were grown from 20% (w/v) PEG3350, 0.2 M KNO3 at 20°C using the sitting-drop, vapor-diffusion technique. The crystallization condition for the NAD bound PnpE was 0.1 M Bicine pH 8.5, 20% PEG10000 and 1 mM NAD. Both data of the two kinds of crystals were collected on BL17U beam line at the Shanghai Synchrotron Radiation Facility (Shanghai, China) using a MAR 225 CCD detector. Crystals were equilibrated in a cryoprotection buffer containing 15% glycerol (v/v) plus reservoir buffer and then flash frozen in a 100K nitrogen stream. The diffraction images were processed with HKL2000 .
Structure determination and refinement
X-ray data collection and refinement statistics
Unique reflections (outer shell)
I/σ (outer shell)
R sym (outer shell)
Resolution range (Å)
Number of reflections (|F| > 0)
Total number of atoms
Bond length (Å)
Bond angles (degree)
Flexible docking of substrate to PnpE
AUTODOCK 4.2  was used to carry out the PnpE-substrate flexible docking. Three out of the nine bonds of the substrate molecule γ-hydroxymuconic semialdehyde were set rotatable. A grid box with sufficient margins (40 × 36 × 36 Å), which enveloped the potential active region of PnpE, was placed to restrain the substrate molecule. This potential active region was implicated by the structure of E.coli L-Lactaldehyde dehydrogenases (PDB code: 2imp) , which is homologous with PnpE. In the active region of PnpE, 9 residues (C281, E247, N149, F150, F154, W157, H275, F447, I282) were assigned as flexible. A genetic search algorithm  was used for the docking and a total of 2,500,000 steps of energy evaluations were performed during the docking. Finally, the first ranked docking result according to the interaction energy was chosen as the result. The flexible docking was performed on a 12-core computer station, which consists of Intel Itanium2 Dual Core running at 1.6 GHz. The average wall clock for one docking run was about 2 hours.
Results and discussion
Overall structure of apo-PnpE
The substrate binding domain consists of a central six-stranded β-sheet (β11 to β16) and six α-helices (α8 to α13), while the cofactor binding domain contains a nine-stranded β-sheet (β1-β4) (β6-β10) and seven α-helices (α1-α7) (Figure 1B).
The four homodimers show high structural similarities. Structure comparisons between AC and other three homodimmers give root mean square deviation (rmsd) values of 0.267 Å, 0.326 Å and 0.214 Å for Cα respectively. The dimer interface consists of β5 (residues F130 to K137), β17 (E469 to N476), loop1 (D117 to F130) and loop2 (V477 to R487) (Figure 1B). Loop1 connects β5 and α3. Loop2 is sited in the C terminal. The interface area of the PnpE monomer for dimerization is 3228.7 Å.
Among all the structure-known homologues of PnpE, the bovine aldehyde dehydrogenase  shares the highest sequence identity (45%) with PnpE. The human aldehyde dehydrogenase  follows close behind with a sequence identity of 44%. The result of sequence alignment indicates these proteins may share similar structures. Structure superimposition shows bovine aldehyde dehydrogenase has a rmsd of 0.925 Å for Cα with PnpE, meanwhile the Cα rmsd between human aldehyde dehydrogenase and PnpE is 0.943 Å.
Structure of PnpE-NAD complex
In the PnpE-NAD complex structure, NAD is situated in a cleft of PnpE. The cofactor binding site is composed of two pockets. The adenosine and the first ribose group of NAD bind in one pocket and the nicotinamide ring in the other. There are several hydrogen bond interactions between PnpE and NAD. The adenine N1A amino group of NAD forms a hydrogen bond with OE1 of Q210 of PnpE. The ribose hydroxyl groups O2B and O3B receive hydrogen bonds from the side chain of K172 and V146, respectively. The diphosphate O2N donates a hydrogen bond to W148. The C4N accepts a hydrogen bond from C281. N7N accepts a hydrogen bond from E247 (Figure 2C).
The PnpE structure of the PnpE-NAD complex is quite similar to apo-PnpE with a Cα rmsd of 0.52 Å. The most significant structural discrimination is observed at the loop between L246 and G252, in which the spatial position of the E247 moved 4.87 Å. This segment belongs to NAD binding region. In the apo-PnpE structure, this loop blocks the NAD binding site. In the complex structure, however, it moves to open the binding site to create enough space for NAD binding (Figure 2C).
Structural bases for the specificity of cofactor binding
Molecular docking results and mutagenesis analysis
The atomic coordinates and structure factors have been deposited under the accession codes: 4GO3 and 4GO4 in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
We thank the staff at beam line BL17U at the Shanghai Synchrotron Radiation Facility for support with data collection. This work was supported by the National Natural Science Foundation of China (31070655) to L. Gu.
- Kohring S, Wiegel J, Mayer F: Subunit composition and glycosidic Activities of the Cellulase Complex from Clostridium thermocellum JW20. Appl Environ Microbiol 1990, 56(12):3798–3804.PubMed CentralPubMedGoogle Scholar
- Hotchkiss SA, Hewitt P, Caldwell J, Chen WL, Rowe RR: Percutaneous absorption of nicotinic acid, phenol, benzoic acid and triclopyr butoxyethyl ester through rat and human skin in vitro: further validation of an in vitro model by comparison with in vivo data. Food Chem Toxicol 1992, 30(10):891–899. 10.1016/0278-6915(92)90056-QView ArticlePubMedGoogle Scholar
- Munnecke DM, Hsieh DP: Microbial decontamination of parathion and p-nitrophenol in aqueous media. Appl Microbiol 1974, 28(2):212–217.PubMed CentralPubMedGoogle Scholar
- Ramadan MA: A variable response of degrading bacteria to phosphorus added to natural water. J Appl Bacteriol 1994, 76(4):314–319. 10.1111/j.1365-2672.1994.tb01634.xView ArticlePubMedGoogle Scholar
- Thouand G, Friant P, Bois F, Cartier A, Maul A, Block JC: Bacterial inoculum density and probability of para-nitrophenol biodegradability test response. Ecotoxicol Environ Saf 1995, 30(3):274–282. 10.1006/eesa.1995.1031View ArticlePubMedGoogle Scholar
- Spain JC, Gibson DT: Pathway for Biodegradation of p-Nitrophenol in a Moraxella sp. Appl Environ Microbiol 1991, 57(3):812–819.PubMed CentralPubMedGoogle Scholar
- Spain JC, Wyss O, Gibson DT: Enzymatic oxidation of p-nitrophenol. Biochem Biophys Res Commun 1979, 88(2):634–641. 10.1016/0006-291X(79)92095-3View ArticlePubMedGoogle Scholar
- Jain RK, Dreisbach JH, Spain JC: Biodegradation of p-nitrophenol via 1,2,4-benzenetriol by an Arthrobacter sp. Appl Environ Microbiol 1994, 60(8):3030–3032.PubMed CentralPubMedGoogle Scholar
- Kitagawa W, Kimura N, Kamagata Y: A novel p-nitrophenol degradation gene cluster from a gram-positive bacterium, Rhodococcus opacus SAO101. J Bacteriol 2004, 186(15):4894–4902. 10.1128/JB.186.15.4894-4902.2004PubMed CentralView ArticlePubMedGoogle Scholar
- Chen Y, Zhang X, Liu H, Wang Y, Xia X: Study on Pseudomonas sp. WBC-3 capable of complete degradation of methylparathion. Wei Sheng Wu Xue Bao 2002, 42: 490–497.PubMedGoogle Scholar
- Zhang J-J, Liu H, Xiao Y, Zhang X-E, Zhou N-Y: Identification and characterization of catabolic para-Nitrophenol 4-Monooxygenase and para-Benzoquinone Reductase from Pseudomonas sp. Strain WBC-3. Jounal of Bacteriology 2009, 191(8):2703–2710. 10.1128/JB.01566-08View ArticleGoogle Scholar
- Liu W, Shen W, Zhao X, Cao H, Cui Z: Expression, purification, crystallization and preliminary X-ray analysis of para-nitrophenol 4-monooxygenase from Pseudomonas putida DLL-E4 Acta Crystallographica Section F Structural Biology and Crystallization. Communications 2009, 65: 1004–1006.Google Scholar
- Sophos NA, Vasiliou V: Aldehyde dehydrogenase gene superfamily: the 2002 update. Chem Biol Interact 2003, 143–144: 5–22.View ArticlePubMedGoogle Scholar
- Hempel J, Nicholas H, Lindahl R: Aldehyde dehydrogenases: widespread structural and functional diversity within a shared framework. Protein Sci 1993, 2(11):1890–1900. 10.1002/pro.5560021111PubMed CentralView ArticlePubMedGoogle Scholar
- Ziegler TL, Vasiliou V: Aldehyde dehydrogenase gene superfamily. The 1998 update. Adv Exp Med Biol 1999, 463: 255–263.View ArticlePubMedGoogle Scholar
- Steinmetz CG, Xie P, Weiner H, Hurley TD: Structure of mitochondrial aldehyde dehydrogenase: the genetic component of ethanol aversion. Structure 1997, 5(5):701–711. 10.1016/S0969-2126(97)00224-4View ArticlePubMedGoogle Scholar
- Moore SA, Baker HM, Blythe TJ, Kitson KE, Kitson TM, Baker EN: Sheep liver cytosolic aldehydedehydrogenase: the structure reveals the basis for the retinal specificity of class 1 aldehydedehydrogenases. Structure 1998, 6: 1541–1551. 10.1016/S0969-2126(98)00152-XView ArticlePubMedGoogle Scholar
- Cobessi D, Tete-Favier F, Marchal S, Azza S, Branlant G, Aubry A: Apo and holo crystal structure of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans. J Mol Biol 1999, 290: 161–173. 10.1006/jmbi.1999.2853View ArticlePubMedGoogle Scholar
- Cobessi D, Tete-Favier F, Marchal S, Branlant G, Aubry A: Structural and biochemical investigations of the catalytic mechanism of an NADP dependent aldehyde dehydrogenase from Streptococcus mutans. J Mol Biol 2000, 300: 141–152. 10.1006/jmbi.2000.3824View ArticlePubMedGoogle Scholar
- Perez-Miller SJ, Hurley TD: Coenzyme isomerization is integral to catalysis in aldehyde dehydrogenase. Biochemistry 2003, 42(23):7100–7109. 10.1021/bi034182wView ArticlePubMedGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T (Eds): Molecular cloning: a laboratory manual. 2nd edition. NY: Cold Spring Harbor Laboratory Press; 1989.Google Scholar
- Herlitze S, Kuenen M: A general and rapid mutagenesis method using polymerase chain reaction. Gene 1990, 91: 143–147. 10.1016/0378-1119(90)90177-SView ArticlePubMedGoogle Scholar
- Otwinowski Z, Minor W: Macromolecular crystallography. Methods Enzymol 1997, 276: 307–326.View ArticleGoogle Scholar
- Winn MD: Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr 2011, D67: 760–763.Google Scholar
- Adams PD, Grosse-Kunstleve RW, Hung LW, Ioerger TR, McCoy AJ, Moriarty NW, Read RJ, Sacchettini JC, Sauter NK, Terwilliger TC: Phenix: building new software for automated crystallographic structure determination. Acta Crystallogr D Biol Crystallogr 2002, 58: 1948–1954. 10.1107/S0907444902016657View ArticlePubMedGoogle Scholar
- Emsley P, Cowtan K: Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 2004, 60: 2126–2132. 10.1107/S0907444904019158View ArticlePubMedGoogle Scholar
- MORRIS GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK, Olson AJ: Automated docking using a Lamarckian genetic algorithm and empirical binding free energy function. J Computational Chemistry 1998, 19: 1639–1662. 10.1002/(SICI)1096-987X(19981115)19:14<1639::AID-JCC10>3.0.CO;2-BView ArticleGoogle Scholar
- Costanzol D, Gomez GA, Christianson DW: Crystal structure of lactaldehyde dehydrogenase from Escherichia coli and inferences regarding substrate and cofactor specificity. J Mol Biol 2007, 366(2):481–493. 10.1016/j.jmb.2006.11.023View ArticleGoogle Scholar
- Gonzalez-Segura L, Rudino-Pinera E, Munoz-Clares RA, Horjales E: The crystal structure of a ternary complex of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa provides new insight into the reaction mechanism and shows a novel binding mode of the 2′-phosphate of NADP + and a novel cation binding site. J Mol Biol 2009, 385(2):542–557. 10.1016/j.jmb.2008.10.082View ArticlePubMedGoogle Scholar
- Ni L, Zhou J, Huriev TD, Weiner H: Human liver mitochondrial aldehyde dehydrogenase: three-dimensional structure and the restoration of solubility and activity of chimeric forms. Protein Sci 1999, 8(12):2784–2790.PubMed CentralView ArticlePubMedGoogle Scholar
- Tsybovsky Y, Donato H, Krupenko NI, Davies C, Krupenko S: A: crystal structure of the C-terminal domain of rat 10’formyltetrahydrofolate dehydrogenase in complex with NADP. Biochemistry 2007, 46: 2917–2929. 10.1021/bi0619573View ArticlePubMedGoogle Scholar
- Sheikh S, Ni L, Hurley TD, Weiner H: The potential roles of the conserved amino acids in human liver mitochondrial aldehyde dehydrogenase. J Biol Chem 1997, 272(30):18817–18822. 10.1074/jbc.272.30.18817View ArticlePubMedGoogle Scholar
- Farres J, Wang TT, Cunningham SJ, Weiner H: Investigation of the active site cysteine residue of rat liver mitochondrial aldehyde dehydrogenase by site-directed mutagenesis. Biochemistry 1995, 34(8):2592–2598. 10.1021/bi00008a025View ArticlePubMedGoogle Scholar
- Wang X, Weiner H: Involvement of glutamate 268 in the active site of human liver mitochondrial aldehyde dehydrogenase as probed by site-directed mutagenesis. Biochemistry 1995, 34: 237–243. 10.1021/bi00001a028View ArticlePubMedGoogle Scholar
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