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

Article abstract of DOI:10.1021/acs.biochem.8b00532

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.

Full text of DOI:10.1021/acs.biochem.8b00532

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Article
Structural Characterization of the Hydratase-Aldolases, NahE
and PhdJ: Implications for Specificity, Catalysis, and the N-
acetylneuraminate 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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Structural Characterization of the HydrataseꢀAldolases, NahE and PhdJ: Implications for
Specificity, Catalysis, and the Nꢀacetylneuraminate Lyase Subgroup of the Aldolase
Superfamily
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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
*Coꢀcorresponding authors
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ABSTRACT
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NahE and PhdJ are bifunctional hydrataseꢀaldolases 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
hydrataseꢀaldolases catalyze a major step in the PAH degradative pathways, their
mechanisms are poorly understood. Sequence analysis identified NahE and PhdJ as
members of the Nꢀacetylneuraminate 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 Asnꢀ281/Gluꢀ285 and Serꢀ278/Aspꢀ282. Mutagenesis is consistent with
the latter. Finally, the PhdJ complex structure suggests a potential mechanism for
hydration of substrate and subsequent retroꢀaldol 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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INTRODUCTION
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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).
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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 hydrataseꢀaldolase. In the naphthalene catabolic pathway, transꢀoꢀ
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hydroxybenzylidenepyruvate hydrataseꢀaldolase (designated NahE) converts transꢀoꢀ
hydroxybenzylidenepyruvate (5, Scheme 2) to salicyaldehyde (9) and pyruvate (11) (6).
In the phenanthrene catabolic pathway, transꢀoꢀcarboxybenzylidenepyruvate hydrataseꢀ
aldolase (designated PhdJ), converts oꢀcarboxybenzylidenepyruvate (6, Scheme 2) to 2ꢀ
carboxybenzaldehyde (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 Nꢀacetylneuraminate
lyase (NAL) subgroup members in the Class I aldolase superfamily. Members of this
subfamily show a conserved (β/α)8ꢀbarrel 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 αꢀketo 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 NahEꢀ and PhdJꢀcatalyzed reactions,
this involves binding of the oꢀhydroxyꢀ or oꢀcarboxybenzylidene moiety and the addition
of water at C4 (of 5 and 6, Scheme 2), to set up a retroꢀaldol fission.
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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
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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
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Materials. Chemicals, biochemicals, buffers, and solvents were purchased from
SigmaꢀAldrich Chemical Co. (St. Louis, MO), Fisher Scientific Inc. (Pittsburgh, PA),
Fluka Chemical Corp. (Milwaukee, WI), or EMD Millipore, Inc. (Billerica, MA). 2ꢀ
Carboxybenzaldehyde (10) was obtained from SigmaꢀAldrich Chemical Co. Phenylꢀ
Sepharose 6 Fast Flow resin was obtained from GE Healthcare Bioꢀsciences (Pittsburgh,
PA). The EconoꢀColumn® chromatography columns were obtained from BioRad
(Hercules, CA). The Amicon stirred cell concentrators and the ultrafiltration membranes
(10,000ꢀDa, MW cutoff) were purchased from EMD Millipore Inc.
Deoxyoligonucleotide primers were synthesized by SigmaꢀAldrich.
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Bacterial Strains and Plasmids. The plasmid designated pRE701 (carrying the nahE
gene) was a gift from Dr. Richard Eaton. The Mycobacterium vanbaalenii PYRꢀ1
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 (ESIꢀMS) was carried out on an LCQ electrospray ionꢀtrap
mass spectrometer (Thermo, San Jose, CA) in the Proteomics Facility in the ICMB.
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Steadyꢀstate kinetic assays were performed on an Agilent 8453 diodeꢀarray
spectrophotometer at 22 °C. Nonꢀlinear 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 sulfateꢀ
polyacrylamide gel electrophoresis (SDSꢀPAGE) 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
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sulfoxide (DMSO)ꢀd6) as the lock signal, except for the C 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 ꢀL DMSOꢀ
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d6, 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 oꢀ
carboxybenzaldehyde (12, Scheme 3) was synthesized by mixing oꢀcarboxybenzaldehyde
(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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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 Gꢀ25 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
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product (as the disodium salt). 6: H NMR (100 mM NaH2PO4, pH 7, 30 ꢀL DMSOꢀd6,
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 =
13
16.3); 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
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following a literature procedure (26). 16: H NMR (100 mM NaH2PO4, pH 7, 30 ꢀL
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
polymerase)
and
with
5'ꢀ
5'ꢀ
TAGTAGTAGCATATGTTGAATAAAGꢀ3'
GATGATGATCTCGAGTCATTATTATTTACTGTATTTAGCGTGꢀ3', 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 SiteꢀDirected Mutagenesis Kit (Agilent Technologies) following
the manufacturer's instructions.
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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 ꢁg/mL), and gentamicin
(20 ꢁg/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 2ꢀL
Erlenmeyer flasks containing 500 mL of M9 minimal media with 0.4% glucose,
kanamycin (50 ꢁg/mL), and gentamicin (20 ꢁg/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 βꢀDꢀthiogalactoside
(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.
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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 Wꢀ385
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
[15ꢀ0% 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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gradient was used (50 mM NaH2PO4 buffer to deꢀionized water), which resulted in
complete elution of protein. Fractions (150 drops) were collected and analyzed by SDSꢀ
PAGE for the appearance of a band (~35 kDa). Fractions were pooled based on the purity
(assessed by SDSꢀPAGE). 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 (0ꢀ0.5 M NaCl in the NaH2PO4 buffer, ~100 mL). Fractions (~2 mL) were
collected and pooled based on purity (as assessed by SDSꢀPAGE) and dialyzed into 50
mM NaH2PO4 buffer (pH 7.0).
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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 SDSꢀPAGE, and pooled based on purity. The pooled
fractions were concentrated and divided into small aliquots. These stocks were flashꢀ
frozen 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 SDSꢀPAGE and electrospray ionization mass spectroscopy (ESIꢀ
MS).
Preparation of SeMet-NahE. The Lꢀselenomethionine (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 0.5ꢀ
1.0. 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 wildꢀtype except
dithiothreitol (5 mM) was added to all buffers to prevent oxidation of selenium. The sizeꢀ
exclusion chromatography step was included in the purification.
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Construction of the Expression Vectors for Native and Mutant PhdJ. The gene, phdJ
(Mvan_0469), was amplified from the M. vanbaalennii PYRꢀ1 genomic DNA using the
PCR (with Taq polymerase) with 5'ꢀCGAGAGAGCATATGGTGCACGTꢀ3' and 5'ꢀ
TCCTCAGGATCCGTGGTTCGAGACꢀ3' 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 Siteꢀ
Directed 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 PYRꢀ1. As a result, the expressed proteins had an additional valine on the
Nꢀterminus.
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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recommended by the manufacturer. The cultures were grown at 37°C overnight.
Subsequently, 4 2ꢀL Erlenmeyer flasks containing 400 mL ZYMꢀ5052 autoꢀinduction
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 (0ꢀ0.5 M NaCl in the NaH2PO4 buffer, 85 mL) Fractions (1.4 mL) were pooled
based on the presence of an SDSꢀPAGE 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 <95% (as assessed by SDSꢀPAGE). Fractions were divided into small aliquots
(50ꢀ200 ꢀL), flashꢀfrozen with liquid nitrogen, and stored at ꢀ80°C.
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PhdJ
Activity
with
trans-o-Hydroxybenzylidenepyruvate
(5),
trans-o-
5
6
7
8
Carboxybenzylidenepyruvate (6), and trans-Benzylidenepyruvate (16). The enzymatic
activities of PhdJ with 5, 6, and 16 were determined as follows. For 5, the substrate (2.7
mg of free acid) was dissolved in ethanol (1 mL). Diluted solutions (1.8, 1.4, 0.90, 0.54,
and 0.36 mg/mL) were made in ethanol using the stock solution. Aliquots (6 ꢀL) of these
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solutions were added to 50 mM NaH2PO4 buffer, pH 7.0 (942 ꢀL) so that the final
concentrations of 5 were 85, 56, 42, 28, 17, and 11 µM. The reactions were initiated by
the addition of PhdJ (50 µL of a 0.51 mg/mL solution to give a final enzyme
concentration of 0.71 µM). Reactions were monitored by following the decrease in
absorbance at 296 nm (9,400 Mꢀ1cmꢀ1). Spectra were recorded for 60 s at 5ꢀs intervals.
The extinction coefficient for 5 was determined by measuring the absorbance of 5 (68
µM) in ethanol (2.5%) in triplicate. The solution was made up by dissolving 5 (5.2 mg) in
ethanol (10 mL) and diluting 25 µL of the stock solution into 50 mM NaH2PO4 buffer,
pH 7.0 (970 µL). Initial rates were calculated using the differences in the extinction
coefficients at 296 nm for 5 (10,700 Mꢀ1 cmꢀ1) and salicylaldehyde (9, 1260 Mꢀ1cmꢀ1).
The extinction coefficient for 9 was determined by dissolving 6 mg in 50 mM NaH2PO4
buffer, pH 7.0 (3 mL). An aliquot (5 µL) was diluted into buffer (995 µL) to make a final
concentration of 82.6 µM. The absorbance at 296 nm was measured in triplicate.
For 6, the substrate (2.0 mg) was added to 50 mM NaH2PO4 buffer, pH 7.0 (500 ꢀL)
to make a 16 mM stock solution (pH ~8). The stock solution was diluted in the same
buffer to make various concentrations (8.1, 4.1, 2.4, 1.6, 1.1, and 0.81 mM).
Subsequently, aliquots (10 µL) of these diluted stock solutions were further diluted into
985 µL of buffer to give final concentrations of 81, 41, 24, 16, 11, and 8.1 µM. The
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reactions were initiated by the addition of PhdJ (5 µL of a 0.51 mg/mL solution to make a
final enzyme concentration of 71 nM). Reactions were monitored by following the
decrease in absorbance at 300 nm (ε = 9,050 Mꢀ1cmꢀ1). Spectra were recorded for 60 s at
3ꢀs intervals. The extinction coefficient for 6 was determined by measuring the
absorbance of a solution of 6 (81 µM) in 50 mM NaH2PO4 buffer, pH 7.0 (in triplicate).
This concentration is the initial absorbance of the highest concentration of 6 before the
addition of enzyme. The initial rates were calculated using the difference between
extinction coefficients at 300 nm (10,500 Mꢀ1cmꢀ1) for 6 and orthoꢀcarboxybenzaldehyde
(10) (1450 Mꢀ1 cmꢀ1). The extinction coefficient for 10 was measured by dissolving 3.6
mg in buffer (1 mL). An aliquot (5 µL) was further diluted in buffer (995 µL) and the
absorbance was measured in triplicate.
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For 16, the substrate (5.4 mg of free acid) was dissolved in ethanol (1 mL). This
solution was diluted with ethanol to make stock solutions of 1.1, 0.72, 0.54, 0.36, 0.27,
0.18, and 0.12 mg/mL. Aliquots (7 µL) of the diluted stock solution were added to 50
mM NaH2PO4 buffer, pH 7.0 (943 µL) so that the final concentrations of 16 were 48, 32,
24, 16, 12, 7.9, and 5.3 µM. The reactions were initiated by the addition of PhdJ (10 µL
of a 0.51 mg/mL solution to give a final concentration of 1.4 µM). Reactions were
monitored by following the decrease in absorbance at 300 nm (20,000 Mꢀ1 cmꢀ1). Spectra
were recorded for 10 min at 20ꢀs intervals. The extinction coefficient for 16 was
determined by measuring the absorbance of a solution made up as follows. A solution of
16 (9.7 mg) in ethanol (1 mL) was diluted 10ꢀfold with ethanol. An aliquot (5 µL) was
added to 50 mM NaH2PO4 buffer, pH 7.0 (1 mL) and the absorbance was measured five
times. Initial rates were calculated using the differences in the extinction coefficients at
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300 nm for 16 (21,000 Mꢀ1 cmꢀ1) and benzaldehyde (540 Mꢀ1 cmꢀ1). The extinction
coefficient for benzaldehyde was determined by diluting a solution of benzaldehyde (4.4
mg in 1 mL ethanol) 200ꢀfold in 50 mM NaH2PO4 buffer, pH 7.0 (1 mL). The absorbance
of the resulting solution was measured in triplicate.
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S278N and D282E PhdJ Activities with 5 and 6. For 5, aliquots (7 ꢀL of 0.30ꢀ2.20
mg/mL stock solutions made up in ethanol) were diluted into the 50 mM NaH2PO4
buffer, pH 7.0 (986 ꢀL) to make final concentrations (11, 16, 23, 36, 54, and 80 ꢀM) of
5. The reactions were initiated by the addition of S278N PhdJ (240 nM) or D282E PhdJ
(310 nM). For 6, aliquots (7 ꢀL of 0.52ꢀ2.75 mg/mL stock solutions) were diluted in 50
mM NaH2PO4 buffer, pH 7.0 (986 ꢀL) to make final concentrations (19, 28, 42, 70, and
100 ꢀM) of 6. The reactions were initiated by the addition of S278N PhdJ (340 nM) or
D282E PhdJ (160 nM). Saturation could not be achieved for 6 so the kcat/Km values were
obtained from a linear fit of the plot of vo vs [S] (28).
1H NMR Identification of the Products in the PhdJ-catalyzed Reaction. Two PhdJꢀ
catalyzed reactions were followed by 1H NMR spectroscopy (using 5 or 6). To an NMR
tube containing 100 mM NaH2PO4 buffer (0.6 mL, pH 7.5) is added a quantity of 5 or 6
[4.5 mg in 30 µL dimethyl sulfoxide (DMSO)ꢀd6)] to reach a final concentration of 30
mM. The final pH of the solution is 7.47. DMSOꢀd6 was used as the lock signal and for
the standardization of the chemical shifts (at 2.49 ppm). The reaction was initiated by
adding PhdJ (100 µL of a 0.51 mg/mL solution) for a final concentration of 1.9 µM.
Spectra were recorded every 3 min over a 33 min period. 9: 1H NMR (H2O, 600 MHz) δ
9.56 (1H, s), 7.5 (1H, dd, J = 1.3 Hz, 7.7 Hz), 7.42 (1H, m), 6.89 (1H, m), 6.81 (1H, d, J
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= 8.4 Hz); 10: H NMR (H2O, 600 MHz) δ 9.80 (1H, s), 7.68 (1H, d, J = 7.7 Hz), 7.52
5
6
7
(1H, m), 7.39 (2H, m); 11: 1H NMR (H2O, 600 MHz) δ 2.17 (3H, s).
8
9
Covalent Modification of PhdJ by 6 in the Presence of NaCNBH3. A reaction mixture
was made up in 50 mM NaH2PO4 buffer (pH 7.0) containing PhdJ (0.5 mg/mL solution)
and NaCNBH3 (15 mg/mL in 100 mM Na2HPO4). The final volume was 120 ꢀL (before
addition of aliquots of 6). The NaCNBH3 solution was made to 0.75 M in 100 Na2HPO4
buffer (pH ~9). Aliquots from a stock solution of 6 (260 µM) were added to the reaction
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mixture (1ꢀ15 ꢀL increments) until enzyme activity had decreased more than 50% (2ꢀ3
h). All reactions were carried out at room temperature. Samples were prepared as
described elsewhere and subjected to ESIꢀMS analysis (29).
Crystallization of the NahE SeMet variant. Initial crystallization conditions for NahE
were identified using sparseꢀmatrix screening with a Phoenix crystallization robotic
system (Art Robbins Instruments) and a protein concentration of 6.5 mg/mL in 50 mM
sodium phosphate buffer at pH 7.0. The conditions that identified initial hits were then
optimized by systematic optimization. The diffractionꢀquality crystals for the
selenomethionine (SeMet) NahE variant were grown in a solution containing 0.1 M 2ꢀ
morpholinꢀ4ꢀylethanesulfonic acid (MES) buffer (pH 6.5ꢀ8.0) and 21ꢀ26% PEG 2000
monomethyl ether (MME) at room temperature.
Crystallization of PhdJ and Soaking with 6. PhdJ at a concentration of 3.4 mg/mL
was used for sparseꢀmatrix crystal screening in the Phoenix crystallization robot (Art
Robbins Instruments). Initial hits were identified and then optimized by systematic
optimization. Diffracting quality crystals were obtained in the sitting drop using vaporꢀ
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diffusion method at room temperature with condition of 0.1 M MES at pH 7ꢀ7.5 and 22ꢀ
25% in PEG 3350.
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The complex crystal of PhdJ with compound 6 was obtained by soaking PhdJ
crystals in a solution of 6 at a concentration of 10 mM for 4 min at room temperature.
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The solution of 6 was made up by diluting a 10× stock solution (1.2 mg of 6 dissolved in
45µL of 100 mM Na2HPO4 buffer, pH 8ꢀ9) into mother liquor at pH 7.0. The crystals of
NahE, PhdJ and PhdJꢀligand complex were cryoꢀprotected by mother liquors containing
25ꢀ30% glycerol before vitrified in liquid nitrogen in preparation for data collection.
Data Collection, Processing, Structure Determination, and Refinement. Xꢀray
diffraction data for the SeMetꢀNahE were collected at Advanced Light Source (ALS)
beamline 8.2.1 (Berkeley, CA) where diffractions in multiple wavelengths were captured. Xꢀ
ray diffraction data of PhdJ and the PhdJꢀsubstrate complex were obtained at Advanced
Light Source beamline 5.0.3 (ALS, Berkeley, CA). Diffraction data were indexed,
integrated, and scaled using HKL2000 (30). Data collection statistics are summarized in
Table 1.
Phases for the SeMetꢀNahE were calculated using Autosol from the PHENIX suite of
programs (31) using multiwavelength anomalous dispersion (MAD). The native PhdJ
structure was determined by molecular replacement (MR) using Phaser (32) and
Autobuild (33) from the PHENIX suite of programs. The monomeric NahE was used as a
search model for the initial estimates of the PhdJ structure factors. Subsequently, the
structure of the native PhdJ was used as a search model for PhdJꢀsubstrate structure
determinations. Structure refinement was carried out using PHENIX Refine (34), in
which TLS parameter refinement was included in the refinement. The structure models
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were evaluated using Molprobity (35) and Procheck (36). The refinement statistics for all
structures are summarized in Table 1. All figures were prepared with PyMol (37).
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Table 1. Crystallographic data and refinement statistics for NahE, PhdJ and PhdJꢀ6
6
7
adduct.
8
9
Data Collection
space group
PhdJ
PhdJꢀ6
P21
NahEꢀSeMet
P3221
P21
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cell dimensions
a, b, c (Å)
52.4, 137.0, 88.3
90.0, 99.1, 90.0
52.5, 137.2, 88.3
90.0, 98.9, 90.0
85.3, 85.3, 133.3
α, β, γ (deg)
90.0, 90.0, 120.0
40.0ꢀ1.94
40.00ꢀ2.05
50.00ꢀ2.00 (2.07ꢀ
(2.01ꢀ1.94)1
resolution (Å)
Rsym
(2.12ꢀ2.05)1
2.00) 1
0.150 (0.506)1
0.996(0.969) 1
15 (4.5)1
0.104 (0.496) 1
0.992(0.68) 1
11 (2.1) 1
0.087 (0.326) 1
0.995(0.872) 1
14 (3.2) 1
CC1/2
I/σI
100.0 (100.0)1
completeness (%)
94.3 (83.6) 1
93.5 (88.7) 1
Refinement
resolution (Å)
39.90ꢀ2.05
72626
45.29ꢀ2.00
77204
38.09ꢀ1.94
42138
no. of unique reflections
Rwork/Rfree (%)
17.95/22.39
16.34/19.20
13.55/18.63
no. of atoms
protein
9893
894
9960
1116
5053
650
water
Average B factor (Å2)
protein
32.9
39.1
23.3
33.7
14.1
26.6
water
rmsd
bond lengths (Å)
bond angles (deg)
Ramachandran plot (%)
most favored regions
allowed regions
disallowed regions
0.002
0.644
0.003
0.772
0.010
1.140
98.2
1.85
0.00
98.3
1.70
0.00
98.3
1.7
0.0
1Data for the last resolution shell are given in parentheses.
2Rfree is calculated with 5% of data randomly omitted from refinement.
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RESULTS AND DISCUSSION
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Dynamic Light Scattering. Two peaks were observed in the LC chromatograph (19.9
min and 22.4 min) for NahE. The first peak (97.5% mass fraction) had a molar mass
moment of 145,700 Da. This species is likely the tetrameric form of NahE (calculated
mass, 146,548 Da). The second peak (2.5% mass fraction) had a molar mass of 36,660
Da and this species is presumed to be monomeric NahE, as the calculated monomeric
mass is 36,637 Da.
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The calculated monomeric mass of PhdJ is 35,966.8 Da. Of the 4 absorbance peaks
observed in the LC chromatogram for a sample, the largest peak (accounting for 88.0% of
the sample mass) corresponded to a PhdJ tetramer (molar mass moment of 138,300). The
fourth peak contained 4.8% of the total mass and is consistent with a trimer of tetramers
(molar mass moment of 421,500). The other two peaks (with 7.2% of the mass of the
sample) are likely impurities from the protein preparation. Thus, the predominant PhdJ
molecule in solution is a tetramer.
Sequence Analysis. An alignment of representative sequences in the Nꢀ
acetylneuraminate lyase (NAL) subgroup was carried out using the program ESPript
(Figure 1) (38). The alignment includes three hydrataseꢀaldolaseꢀcatalyzed reactions in
the bacterial catabolic pathways for phenanthrene (PhdJ and PhdG) and the naphthalene
(NahE) (Scheme 2). (PhdG is proposed to catalyze an earlier step in phenanthrene
catabolism.) The alignment shows the strict conservation of Lysꢀ180 (using the PhdJ
numbering system), Tyrꢀ152, and the GXXGE motif (Glyꢀ61, Thrꢀ62, Pheꢀ63, Glyꢀ64,
Gluꢀ65) at the active site. These residues are the soꢀcalled primary residues involved in
the common reaction of the NAL subgroup, which is Schiff base formation and binding
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of the αꢀketo acid moiety (of substrate). Three other residues are also conserved (Gluꢀ74,
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8
Argꢀ298, Proꢀ300), but these are found outside the active site and might play a structural
role.
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Figure 1. Sequence alignment of representative NAL subgroup members. The secondary
structural elements for PhdJ are shown above the alignment. The αꢀhelices and 310 (η)
helices are indicated by large and small squiggles, respectively. βꢀstrands are shown as
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arrows and βꢀturns are indicated by TT letters. Red letters indicate similar residues and
white lettering with red fill indicates conserved residues. Similar and conserved residues
are shown in blue boxes. The dots beneath residues indicate primary residues (blue) in
the NAL subgroup, secondary residues (red) common in hydrataseꢀaldolases, and
secondary residues (green) that are specific to individual hydrataseꢀaldolases, as
discussed in the text. The red line indicates an insertion in the hydrataseꢀaldolases that
codes for αꢀhelix 10, as discussed in the text. The sequences shown are M. vanbaalenii
PYRꢀ1 PhdJ (GI: 49072891), M. vanbaalenii PYRꢀ1 PhdG (GI: 49072894),
Pseudomonas putida NahE (GI: 483790), Escherichia coli NAL (GI: 216589), E. coli
DHDPS (GI: 758861597), Sulfolobus solfataricus KDGA (GI: 27429444), and
Agrobacterium fabrum KDGDH (GI: 1134942). The alignment was carried out and the
Figure was generated using ESPript version 3.0 (38).
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Three other residues, Asnꢀ154, Trpꢀ225, and Pheꢀ274, are conserved in the three
hydrataseꢀaldolases (indicated by a red dot). Eight additional residues are specific to the
individual hydrataseꢀaldolase (indicated by a green dot). In PhdJ, these residues are Thrꢀ
62, Trpꢀ125, Leuꢀ182, Pheꢀ271, Leuꢀ275, Serꢀ278, Aspꢀ282, and Glnꢀ285. In NahE, the
first four residues are the same, but the last four residues (Leuꢀ275, Serꢀ278, Aspꢀ282,
Glnꢀ285) are replaced by Ser, Asn, Glu, and Arg (respectively). All 11 residues are
likely the soꢀcalled secondary residues that are responsible for the unique chemistry of
the hydrataseꢀaldolases and their specificities.
Covalent Modification of PhdJ in the Presence of Substrate and NaCNBH3. PhdJ was
incubated with substrate (6) in the presence of NaCNBH3 (for 2ꢀ3 h). Activity decreased
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significantly when compared to a control sample without compound or NaCNBH3. The
ESI mass spectrum of the control sample showed a major signal at 35,967 Da whereas the
mass spectrum of the treated sample showed a major signal at 36,173 Da (calculated,
36,169.8 Da). The mass difference, 206 Da, is consistent with the reduced imine of 6
attached to the enzyme. The loss of PhdJ activity (in the presence of NaCNBH3 and 6)
coupled with a single covalent modification is consistent with the formation of a Schiff
base between substrate and enzyme. A similar result for NahE has been previously
reported (39).
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1H NMR Characterization of the PhdJ-catalyzed Reactions. The PhdJꢀcatalyzed
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reaction using 6 was monitored by H NMR spectroscopy in order to confirm the
identities of the products. After 33 min, the spectra show signals consistent with the
formation of 10 and 11 from 6 (Scheme 2). In a similar NMR experiment, the spectra
show signals that indicate the formation of 9 and 11 from 5. The NMR experiments show
that both substrates are processed by PhdJ and afford products that are consistent with the
proposed reactions.
Kinetic Analysis. The activity of PhdJ was measured with three substrates (5, 6, and
ꢀ
16) (Table 2). The substrates vary by the orthoꢀsubstituent on the ring (R = OH, CO2 ,
H). The best substrate is 6, as assessed by the highest kcat/Km value (~8.2 × 104 Mꢀ1 sꢀ1).
The value is consistent with those in the literature and the proposed role of PhdJ in the
catabolic pathway (14). Replacing the orthoꢀcarboxylate group with a hydroxyl group
(i.e., 5, the substrate for NahE) results in 26ꢀfold drop in the kcat/Km value (due mostly to
the 23ꢀfold drop in kcat). Finally, removing the orthoꢀsubstituent entirely (i.e., 16) causes
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a 1025ꢀfold drop in the kcat/Km value. It is also not possible to saturate the enzyme with
16.
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Crystal structure of NahE. In order to gain structural insights into catalysis and
specificity of the hydrataseꢀaldolases, the structures of NahE, PhdJ, and a complex of
PhdJ with 6 were determined. Due to the low sequence similarity between NahE and
other NAL family members, the structure of NahE was solved by de novo phasing using
the SeMet derivative of NahE (19). The final model is refined to 1.9 Å resolution with
two monomers per asymmetric unit (Table 1). As evident from light scattering
experiments, NahE functions as a tetramer in solution. In the crystal structure, NahE
shows a configuration of dimer of dimers with two monomers forming a tight dimer and
then the pair of dimers forming the physiologically functioning unit of a tetramer (Figure
2A). This results in two different dimer interfaces: a tight interface for the two monomers
to form a dimer with 2009.3 Å2 accounting for 17.4% of the surface and a loose interface
between the two dimers with only 746.1 Å2 for 5.4% of surface, as calculated by the
PISA program (40). The overall fold of NahE is typical of the NAL subgroup members
(15,19), which are composed of (α/β)8ꢀbarrels at the Nꢀterminal catalytic moiety of the
enzyme (residue 8ꢀ250), followed by three Cꢀterminal helices tilted on the side of the
barrel (residue 251ꢀ331) (Figure 2B). The active site of NahE is located at the heart of the
barrel with the highly conserved features of the NAL subgroup (Lysꢀ183, Tyrꢀ155, and
the GXXGE motif) (Figure 1 and Figure 2C) (15,19). In addition, hydrophobic residues
(Ileꢀ22, Pheꢀ66, Metꢀ122, Tyrꢀ155, Trpꢀ224) dominate the active site pocket. Trpꢀ128,
from the neighboring monomer, also participates the formation of the active site (Figure
2C).
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Figure 2.
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Figure 2. Overview of the NahE and PhdJ structures. A) NahE tetramer shown with a
different color for each monomer. B) Overall structure of the NahE monomer shown as a
ribbon diagram. The Nꢀterminal TIM barrel domain is shown with the αꢀhelices in wheat,
βꢀstrands in yellow, and coil in green. The Cꢀterminal helical bundle is shown in pink.
The highly conserved active site residues Tyrꢀ155 and Lysꢀ183 are highlighted in carbon
colored in cyan and shown as sticks. C) The active site of NahE. Important residues are
shown as sticks with carbon atoms colored cyan. The residue from the adjacent monomer
is colored in gold. D) PhdJ tetramer shown with a different color for each monomer. E)
Overall structure of PhdJ monomer shown as a ribbon diagram. The Nꢀterminal TIM
barrel domain is shown with the αꢀhelices in cyan, βꢀstrands in violet, and coil in pink.
The Cꢀterminal helical bundle is colored yellow. The highly conserved active site
residues Tyrꢀ152 and Lysꢀ180 are shown as sticks with carbon atoms colored green. F)
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The active site of PhdJ. Important active site residues are shown as sticks with the carbon
atoms colored green. The residue from the adjacent monomer is colored silver.
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Crystal Structure of Native PhdJ. The crystal structure of the native PhdJ was
determined by molecular replacement using the NahE structure as the search model. The
final structure was refined to 2.1 Å resolution (Table 1). Similar to NahE, PhdJ maintains
the same architecture of tetramer formation as a dimer of dimers (Figure 2D), consistent
with dynamic light scattering profile in solution. The dimer in between the monomers is
1983.1 Å2 (accounting for 15.2% of the surface), substantially larger than the interface
between the dimers (635.5 Å2, 4.7% of the surface). For each monomer, a core (α/β)8ꢀ
barrel (TIM barrel) forms the catalytic portion of the enzyme (residue 1ꢀ248) followed by
a Cꢀterminal helical bundle (residue 249ꢀ329) leaning against the barrel (Figure 2E). The
conserved Lysꢀ180, implicated in Schiff base formation and hydrolysis, is found at the
center of the barrel on βꢀstrand 6, and indicates the location of the active site. The
conserved tyrosine (Tyrꢀ154) is found nearby along with the GXXGE motif. Like NahE,
the active site pocket is dominated by hydrophobic residues (e.g., Ileꢀ19, Pheꢀ63, Pheꢀ
119, Tyrꢀ152, Trpꢀ225), suggesting a preference for a hydrophobic substrate (Figure 2F).
Similar to NahE, a conserved tryptophan residue (i.e., Trpꢀ125) from neighboring
monomer forms part of the active site.
Formation of the PhdJ complex with substrate 6. To gain insight into the catalytic
reaction carried out by PhdJ, we soaked the substrate 6 (10 mM) into preꢀformed PhdJ
crystals in mother liquor conditions for 4 min. The crystal data were collected and scaled
to 2.0 Å resolution. The native PhdJ structure was used as the search model to find a
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solution for the structure (Table 1). The folding of the substrateꢀsoaked protein shows an
identical configuration to that of native PhdJ. However, strong positive density is
observed in all four active sites (Figure 3A). The density extends from the side chain of
Lysꢀ180, suggesting the formation of a Schiff base between the side chain of Lysꢀ180 and
the 2ꢀketo group of the substrate. Iterative cycles of ligand building were used to
establish the identity of the ligand at the active site (Figure 3B). Strong and flat positive
density prompted us to model the phenyl ring of 6 in the density. Further calculations
reveal strong positive density connected between the Schiff base (at Cꢀ2) and the phenyl
ring (Figures 3B and 3C). In three of the four active sites, the strong positive density at
the active site is consistent with the formation of a Schiff base between Lysꢀ180 and 6
(Figure 3D). Formation of the Schiff base between PhdJ and substrate is surprising
because the wild type PhdJ is likely still active in the crystalline form. The thermal
factors of the adduct moiety of Lysꢀ180 (~30 Å2) are slightly higher than the main chain
atoms of 20 Å2, but comparable to the overall average B factors of the whole molecule.
We speculate that the short soaking time, high precipitant concentration, and crystal
packing stabilize proteinꢀsubstrate adduct formation and slow down the product turnover
to allow the capture of the complex.
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