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Structural Characterization of the Hydratase-Aldolases, NahE and PhdJ: Implications for Specificity, Catalysis, and the Nacetylneuraminate lyase subgroup of the Aldolase Superfamily Jake LeVieux, Brenda P Medellin, William H. Johnson, Kaci Erwin, Wenzong Li, Ingrid Anita Johnson, Yan Jessie Zhang, and Christian P. Whitman Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00532 • Publication Date (Web): 01 Jun 2018 Downloaded from http://pubs.acs.org on June 1, 2018

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Biochemistry

Structural Characterization of the Hydratase-Aldolases, NahE and PhdJ: Implications for Specificity, Catalysis, and the N-acetylneuraminate Lyase Subgroup of the Aldolase Superfamily

Jake A. LeVieux‡, #, Brenda Medellin‡, #, William H. Johnson, Jr.§, Kaci Erwin‡, Wenzong Li‡, Ingrid A. Johnson, Yan Jessie Zhang‡,$,*, and Christian P. Whitman§,$,*

‡

Department of Molecular Biosciences, §Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, and $Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712

REVISED June 1, 2018

#

These authors contribute equally for the work

*

Co-corresponding authors

ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT NahE and PhdJ are bifunctional hydratase-aldolases in bacterial catabolic pathways for naphthalene and phenanthrene, respectively. Bacterial species with these pathways can use polycyclic aromatic hydrocarbons (PAHs) as sole sources of carbon and energy. Due to the harmful properties of PAHs and their widespread distribution and persistence in the environment, there is great interest in understanding these degradative pathways including the mechanisms and specificities of the enzymes found in the pathways. This knowledge can be used to develop and optimize bioremediation techniques. Although hydratase-aldolases catalyze a major step in the PAH degradative pathways, their mechanisms are poorly understood. Sequence analysis identified NahE and PhdJ as members of the N-acetylneuraminate lyase (NAL) subgroup in the aldolase superfamily. Both have a conserved lysine and tyrosine (for Schiff base formation) as well as a GXXGE motif (to bind the pyruvoyl carboxylate group). Herein, we report the structures of NahE, PhdJ, and PhdJ covalently bound to substrate via a Schiff base. Structural analysis and dynamic light scattering experiments show that both enzymes are tetramers. A hydrophobic helix insert, present in the active sites of NahE and PhdJ, might account for the different properties. The individual specificities of NahE and PhdJ are governed respectively by Asn-281/Glu-285 and Ser-278/Asp-282. Mutagenesis is consistent with the latter. Finally, the PhdJ complex structure suggests a potential mechanism for hydration of substrate and subsequent retro-aldol fission. The combined findings fill a gap in our mechanistic understanding of these enzymes and their place in the NAL subgroup.

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Biochemistry

INTRODUCTION Naphthalene (1, Scheme 1), phenanthrene (2), and other polycyclic aromatic hydrocarbons [e.g., fluoranthene (3), and pyrene (4)] are persistent environmental contaminants that are responsible for many human health problems (1). Their effects can be direct or indirect (via reactive metabolites) (2). Polycyclic aromatic hydrocarbons (PAHs) are also toxic to marine and other aquatic organisms (3,4). Due to all of these adverse effects, there is much interest in the development of technologies for the removal of PAHs from the environment (5).

PAHs can be converted to useful cellular intermediates by bacterial catabolic pathways. The most extensively characterized of these pathways is the one for the degradation of naphthalene in Pseudomonas putida G7 (6). The phenanthrene catabolic pathway is not as well characterized and many proposed enzymatic activities (including the identification of the substrates and products) have not been experimentally verified at the biochemical level. Other reactions have only been partially characterized (7,8). The pathways for the higher molecular weight species (i.e., 3 and 4) are poorly characterized at best (9-13). As part of an effort to use these pathways in bioremediation efforts, we are carrying out a systematic characterization of the individual enzymatic steps. One key reaction in bacterial catabolic pathways for PAHs is catalyzed by a bifunctional hydratase-aldolase. In the naphthalene catabolic pathway, trans-o-

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hydroxybenzylidenepyruvate hydratase-aldolase (designated NahE) converts trans-ohydroxybenzylidenepyruvate (5, Scheme 2) to salicyaldehyde (9) and pyruvate (11) (6). In the phenanthrene catabolic pathway, trans-o-carboxybenzylidenepyruvate hydratasealdolase (designated PhdJ), converts o-carboxybenzylidenepyruvate (6, Scheme 2) to 2carboxybenzaldehyde (10) and pyruvate (8) (8,14). These enzymes process their respective substrates through the putative intermediates 7 or 8 (or the Schiff bases of 7 or 8, and 11) (8,14). Sequence analysis identified NahE and PhdJ as N-acetylneuraminate lyase (NAL) subgroup members in the Class I aldolase superfamily. Members of this subfamily show a conserved (β/α)8-barrel structure, two strictly conserved active site residues (tyrosine and lysine) that are involved in Schiff base formation, and a GXXGE motif that is associated with the binding of the α-keto acid moiety (15-20). Much of the remaining portion of the active site is tailored to accommodate the individual reactions catalyzed by the NAL subfamily members. For the NahE- and PhdJ-catalyzed reactions, this involves binding of the o-hydroxy- or o-carboxybenzylidene moiety and the addition of water at C4 (of 5 and 6, Scheme 2), to set up a retro-aldol fission.

Herein, we report the structural characterization of NahE and PhdJ. The structures were determined by de novo phasing and molecular replacement to resolutions at 1.9 and 2.0 Å, respectively. Examination of a complex structure of PhdJ covalently linked to its substrate (i.e., 6) provides a structural basis for its substrate specificity when compared to 4


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Biochemistry

that of NahE. Mutagenesis supports the explanation. Comparison of the structures identifies elements that differentiate NahE and PhdJ from the other NAL family members. Finally, a conserved water molecule near C4 of the substrate (see 6 in Scheme 2) suggests a reaction mechanism for the addition of water at C4.

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EXPERIMENTAL PROCEDURES Materials. Chemicals, biochemicals, buffers, and solvents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO), Fisher Scientific Inc. (Pittsburgh, PA), Fluka Chemical Corp. (Milwaukee, WI), or EMD Millipore, Inc. (Billerica, MA). 2Carboxybenzaldehyde (10) was obtained from Sigma-Aldrich Chemical Co. PhenylSepharose 6 Fast Flow resin was obtained from GE Healthcare Bio-sciences (Pittsburgh, PA). The Econo-Column® chromatography columns were obtained from BioRad (Hercules, CA). The Amicon stirred cell concentrators and the ultrafiltration membranes (10,000-Da,

MW

cutoff)

were

purchased

from

EMD

Millipore

Inc.

Deoxyoligonucleotide primers were synthesized by Sigma-Aldrich. Bacterial Strains and Plasmids. The plasmid designated pRE701 (carrying the nahE gene) was a gift from Dr. Richard Eaton. The Mycobacterium vanbaalenii PYR-1 genomic DNA was a gift from Dr. Carl Cerniglia (National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR). Escherichia coli ArcticExpress cells were obtained from Agilent Technologies (Santa Clara, CA). General Methods. The PCR amplification of DNA sequences was conducted in a GeneAmp 2700 thermocycler (Applied Biosystems, Carlsbad, CA). Techniques for restriction enzyme digestion, ligation, transformation, and other standard molecular biology manipulations were based on methods described elsewhere (21). DNA sequencing was performed in the DNA Sequencing Facility in the Institute for Cellular and Molecular Biology (ICMB) at the University of Texas at Austin. Electrospray ionization mass spectrometry (ESI-MS) was carried out on an LCQ electrospray ion-trap mass spectrometer (Thermo, San Jose, CA) in the Proteomics Facility in the ICMB.

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Biochemistry

Steady-state kinetic assays were performed on an Agilent 8453 diode-array spectrophotometer at 22 °C. Non-linear regression data analysis was performed using the program Mathematica (Wolfram Research, Inc., Version 8.0, Champaign, Il.) Protein concentrations were determined by the Waddell method (22). Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) was carried out on denaturing gels containing 12% polyacrylamide (23). Nuclear magnetic resonance (NMR) spectra were recorded on a Varian DirectDrive 600 MHz spectrometer (Palo Alto, CA). All NMR spectra were carried out in 100 mM NaH2PO4 buffer (at the indicated pH) using dimethyl sulfoxide (DMSO)-d6) as the lock signal, except for the 13C NMR spectrum of 5, which was carried out in CD3OD. NMR signals were analyzed using the software program SpinWorks 3.1.6 (Copyright © 2009 Kirk Marat, University of Manitoba). The sequence alignments and secondary information were visualized using ESPript version 3.0. The dynamic light scattering experiments were carried out as described elsewhere (24).

Synthesis of trans-o-Hydroxybenzylidenepyruvate (5). A mixture of salicylaldehyde (9, 1 g, 13 mmol), ethyl triphenylphosphoranylidenepyruvate (13) (5 g, 13 mmol), and a catalytic amount of benzoic acid (~50 mg) was heated in anhydrous DMF (2.5 mL) under argon at 80°C, as described previously (25), to yield 14 (Scheme 3). After 15 h, water (25 mL) was added to the reaction mixture and it was extracted with hexanes/ethyl acetate (2:1). The organic layers were combined, dried over anhydrous Na2SO4, passed

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through a small silica plug column, and evaporated to dryness in vacuo. The residue was purified by flash column chromatography (4:1 hexanes/ethyl acetate). Fractions containing 14 were combined, dried over anhydrous Na2SO4, and evaporated to dryness under reduced pressure. The compound was suspended in H2O (5 mL) and the ester was hydrolyzed by the dropwise addition of 1M NaOH (and subsequent ethyl acetate extraction) without exceeding pH 10. The aqueous phase was then rapidly acidified to pH 2 (with 8.5% phosphoric acid) and the product was isolated by extraction with ethyl acetate. The organic layers were collected, dried over anhydrous Na2SO4, and evaporated to dryness to yield ~1 g of product. 5: 1H NMR (100 mM NaH2PO4, pH 7, 30 µL DMSOd6, 600 MHz) δ 6.77 (1H, d, J = 16.5 Hz), 6.80 (2H, m), 7.20 (1H, m), 7.47 (1H, dd, J = 7.8 Hz, 1.5 Hz), 7.79 (1H, d, J = 16.5 Hz); 13C NMR (CD3OD, 125 MHz) δ 117.5, 121.2, 121.7, 122.8, 130.9, 134.4, 146.1, 159.7, 166.2, 187.5 ppm. Synthesis of trans-o-Carboxybenzylidenepyruvate (6). The methyl ester of ocarboxybenzaldehyde (12, Scheme 3) was synthesized by mixing o-carboxybenzaldehyde (o-CBA, 10, 1.5 g, 10 mM) and dimethyl sulfate (1.9 g, 15 mM) with 2 eq of K2CO3. The reaction mixture was stirred at a gentle reflux in acetone (50 mL) overnight. After most of the acetone was removed under reduced pressure, the residue was diluted with ethyl acetate (~100 mL) and filtered to removed salts. The ethyl acetate was washed with water, the organic layer was collected, dried over anhydrous Na2SO4, and evaporated to dryness. For additional purity, the residue was purified by flash column chromatography (4:1 hexanes/ethyl acetate). Fractions containing 12 were combined, dried over anhydrous Na2SO4, and evaporated to dryness.

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Biochemistry

Subsequently, 12 (1g, 13 mmol) was treated with 13 (25), and processed as described above, to form 15. After the flash chromatography step and evaporation of the ethyl acetate, the compound was suspended in H2O (5 mL) and the esters hydrolyzed by the dropwise addition of 1 M NaOH without exceeding pH 10. If necessary, the pH was adjusted to ~8, concentrated to ~2 mL in vacuo, and passed through a G-25 gel filtration column (to desalt). Product (6) was eluted using H2O and identified by UV. Fractions containing 6 were combined and concentrated under reduced pressure to yield ~1 g of product (as the disodium salt). 6: 1H NMR (100 mM NaH2PO4, pH 7, 30 µL DMSO-d6, 600 MHz) δ 6.72 (1H, d, J = 16.3 Hz), 7.31(3H, m), 7.62 (1H, d, 7.4 Hz), 7.86 (1H, d, J = 16.3);

13

C NMR (125 MHz) δ 125.1, 128.7, 129.0, 130.8, 132.1, 132.7, 142.6, 149.7,

173.4, 178.3, 198.6 ppm. Synthesis of trans-Benzylidenepyruvate (16). The compound was synthesized following a literature procedure (26). 16: 1H NMR (100 mM NaH2PO4, pH 7, 30 µL DMSO, 600 MHz) δ 6.72 (1H, d, J = 16.5 Hz), 7.32 (3H, m), 7.52 (3H, m). Construction of the NahE Expression Vector. NahE was amplified from the pRE701 plasmid

using

the

PCR

(and

Taq

TAGTAGTAGCATATGTTGAATAAAG-3'

polymerase) and

with

5'5'-

GATGATGATCTCGAGTCATTATTATTTACTGTATTTAGCGTG-3', as forward and reverse primers, respectively. The primers contained the XhoI and NdeI restriction sites (underlined). The PCR product and the pET24 vector were treated with the appropriate restriction enzymes, ligated, and processed to construct an expression vector for NahE. The PCR introduced three mutations, which were corrected (D265G, S275T, and P299S)

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using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) following the manufacturer's instructions. Expression and Purification of NahE. E. coli ArcticExpress cells (Agilent Technologies) were transformed with the pET vector containing the gene for NahE and used to inoculate LB media (450 mL) containing kanamycin (50 µg/mL), and gentamicin (20 µg/mL). The latter is recommended by the manufacturer. After the cells had been shaken overnight at 37°C, 25 mL of the culture was used to inoculate each of 9 2-L Erlenmeyer flasks containing 500 mL of M9 minimal media with 0.4% glucose, kanamycin (50 µg/mL), and gentamicin (20 µg/mL) (21). The cells were allowed to grow until the OD600 reached ~0.5 (~3 h). In one preparation, the flasks containing the cells were put on ice for 30 min, followed by the addition of isopropyl β-D-thiogalactoside (IPTG) to make each one 1 mM in IPTG. After growing for ~65 h at 15 °C, cells were harvested by centrifugation (15,300 g for 20 min) and stored at -20 °C. In a typical procedure, cells were thawed and suspended in 50 mM NaH2PO4 buffer (pH 7.0, 150 mL) that contained 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM EDTA, and 1.5 mg/mL lysozyme. Cells were lysed by sonication using a W-385 ultrasonicator made by Heat Systems (15 min at 30% duty, 5 s intervals). Ground (NH4)2SO4 was added to the lysate (30% saturation) before centrifugation (39000 g, 30 min). The supernatant was loaded onto a Phenyl Sepharose 6 (~55 mL) column equilibrated in 50 mM NaH2PO4 buffer (pH 7.5) made 15% in (NH4)2SO4. After the column was washed (>1 column volume), protein was eluted by using a linear gradient [15-0% in (NH4)2SO4 in the NaH2PO4 buffer, 200 mL]. The protein did not completely elute from the column, as indicated by a protein determination assay, so a 100 mL linear

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Biochemistry

gradient was used (50 mM NaH2PO4 buffer to de-ionized water), which resulted in complete elution of protein. Fractions (150 drops) were collected and analyzed by SDSPAGE for the appearance of a band (~35 kDa). Fractions were pooled based on the purity (assessed by SDS-PAGE). The protein was dialyzed into 50 mM NaH2PO4 buffer (pH 7.0) overnight. The dialyzed protein was loaded on a DEAE anion exchange column (~30 mL) equilibrated in 50 mM NaH2PO4 buffer (pH 7.0). Protein was eluted using a linear gradient (0-0.5 M NaCl in the NaH2PO4 buffer, ~100 mL). Fractions (~2 mL) were collected and pooled based on purity (as assessed by SDS-PAGE) and dialyzed into 50 mM NaH2PO4 buffer (pH 7.0). Protein used for crystallography was concentrated and further purified by gel filtration chromatography using a HiLoad 16/60 Superdex (120 mL) column connected to a fast protein liquid chromatography (FPLC) system. The protein was eluted isocratically (1 mL/min) in 25 mM HEPES buffer (pH 7.5) made 0.1 M in NaCl. Fractions (1.0 mL) were collected, analyzed by SDS-PAGE, and pooled based on purity. The pooled fractions were concentrated and divided into small aliquots. These stocks were flashfrozen with liquid nitrogen, then stored at -80 °C. Typically the yield for this procedure is ~30 mg per L of culture or 3 mg per g of cells. The purity of NahE was determined to be >95% as assessed by SDS-PAGE and electrospray ionization mass spectroscopy (ESIMS). Preparation of SeMet-NahE. The L-selenomethionine (SeMet) derivative of NahE was expressed and purified similar to wild type enzyme with the following modifications. Cells were grown in M9 minimal media with 0.2% glucose until the OD600 reached 0.51.0. To each flask were added lysine, phenylalanine, threonine (100 mg each); isoleucine,

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leucine, and valine (50 mg each); and selenomethionine (60 mg). Additionally, IPTG (1 mM) was added prior to incubation (16 h) at room temperature. The remainder of the purification protocol for the derivative was identical to that of wild-type except dithiothreitol (5 mM) was added to all buffers to prevent oxidation of selenium. The sizeexclusion chromatography step was included in the purification. Construction of the Expression Vectors for Native and Mutant PhdJ. The gene, phdJ (Mvan_0469), was amplified from the M. vanbaalennii PYR-1 genomic DNA using the PCR (with Taq polymerase) with 5'-CGAGAGAGCATATGGTGCACGT-3' and 5'TCCTCAGGATCCGTGGTTCGAGAC-3' as forward and reverse primers, respectively. The primers contained NdeI and BamHI restriction sites (underlined). The PCR product and the pET24 vector were treated with the appropriate restriction enzymes, ligated, and processed to construct the PhdJ expression vector. The PCR introduced three mutations, which were corrected (K44T, Q325E, and G335Stop) using the QuikChange SiteDirected Mutagenesis Kit following the manufacturer's instructions. The S278N and D282E mutants of PhdJ were constructed using the same kit and the native PhdJ expression vector as the template. E. coli ArcticExpress cells were transformed with the plasmids, following the manufacturer's protocol. In all three plasmids, the ATG start codon is placed immediately before the GUG start codon found in the phdJ gene in M. vanbaalennii PYR-1. As a result, the expressed proteins had an additional valine on the N-terminus. Expression and Purification of Native and Mutant PhdJ. The E. coli ArcticExpress cells (containing the expression vector) were used to inoculate a starter culture with 50 mL LB media with kanamycin (Kn, 50 µg/mL) and gentamicin (20 µg/mL), as

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Biochemistry

recommended by the manufacturer. The cultures were grown at 37°C overnight. Subsequently, 4 2-L Erlenmeyer flasks containing 400 mL ZYM-5052 auto-induction media (27) and Kn (100 µg/mL) were inoculated with 25 mL starter culture (each) and grown until the OD600 reading reached about 0.3 (~3 h). The cultures were then cooled to 12° C and shaken at 250 rpm for 65 h. Cells were harvested by centrifugation (15300 g, 20 min) and stored at -20 °C. The cell pellet mass for 1.6 L of culture was 25.5 g. Cells were lysed in 50 mM NaH2PO4 buffer (pH 7.0, 80 mL), as described above. The lysate was centrifuged (15300 g, 30 min), the pellet discarded, and 30 mL of 50 mM NaH2PO4 buffer, pH 7.0, was added to the supernatant. The resulting solution was then centrifuged again (39000 g for 30 min). The supernatant was loaded onto a DEAE column (17 mL) equilibrated with 50 mM NaH2PO4 buffer, pH 7.0. The column was washed (>1 column volume of the same buffer) and the protein was eluted with a linear gradient (0-0.5 M NaCl in the NaH2PO4 buffer, 85 mL) Fractions (1.4 mL) were pooled based on the presence of an SDS-PAGE band at ~35 kD and activity (with 6). Ammonium sulfate (to 30% saturation) was added slowly to the pooled fractions and stirred for 20 min on ice before centrifugation (39000 g, 20 min). Ammonium sulfate was then added again to the supernatant to reach 40% saturation. After stirring for 20 min on ice, the solution was centrifuged (39000 g, 20 min). The pellet was dissolved into 50 mM NaH2PO4 buffer, pH 7.0. A typical preparation yields ~4.9 mg of purified protein/L culture. An additional chromatography step was performed with Phenyl Sepharose when purity was

REVISED June 1, 2018

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