We have concluded that the removal of the regulatory domain is sufficient to eliminate serine inhibition but does not have the expected effect on the quaternary structure.. Fax: + 1 612 625
Trang 1De-regulation of D -3-phosphoglycerate dehydrogenase by domain removal
Jessica K Bell1, Paul J Pease1, J Ellis Bell2, Gregory A Grant3and Leonard J Banaszak1
1
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA;
2
Department of Chemistry, University of Richmond, Richmond, Virginia, USA;3Department of Molecular Biology
and Pharmacology and the Department of Medicine, Washington University, St Louis, MO, USA
Escherichia coli 3-phosphoglycerate dehydrogenase
(PGDH) catalyzes the first step in serine biosynthesis, and is
allosterically inhibited by serine Structural studies revealed a
homotetramer in which the quaternary arrangement of
subunits formed an elongated ellipsoid Each subunit
consisted of three domains: nucleotide, substrate and
regu-latory In PGDH, extensive interactions are formed between
nucleotide binding domains A second subunit–subunit
interaction occurs between regulatory domains creating an
extended b sheet The serine-binding sites overlap this
interface In these studies, the nucleotide and substrate
domains (NSDs) were subcloned to identify changes in both
catalytic and physical properties upon removal of a subunit–
subunit interface The NSDs did not vary significantly from
PGDH with respect to kinetic parameters with the exception
that serine no longer had an effect on catalysis Temperature
dependent dynamic light scattering (DLS) revealed the
NSDs aggregated > 5C before PGDH, indicating de-creased stability DLS and gel filtration studies showed that the truncated enzyme formed a tetramer This result negated the hypothesis that the removal of the regulatory domain would create an enzyme mimic of the unregulated, closely related dimeric enzymes Expression of the regulatory do-main, to study conformational changes induced by serine binding, yielded a product that by CD spectra contained stable secondary structure DLS and pulsed field gradient NMR studies of the regulatory domain showed the presence
of higher oligomers instead of the predicted dimer We have concluded that the removal of the regulatory domain is sufficient to eliminate serine inhibition but does not have the expected effect on the quaternary structure
Keywords: domains; enzyme regulation; oxidoreductase; 3-phosphoglycerate dehydrogenase; truncation
D-3-Phosphoglycerate dehydrogenase (PGDH) catalyzes
the first committed step in the phosphorylated serine
biosynthetic pathway During the PGDH reaction,
3-phos-phoglycerate (GriP), a glycolytic intermediate, is oxidized to
3-phosphohydroxypyruvate (PHP) with the concomitant
reduction of NAD The pathway, as a branch point off the
glycolysis pathway, is tightly regulated In prokaryotes and
lower plants, an inhibitory feedback loop utilizes serine to
allosterically regulate the initial step of the pathway, the
PGDH reaction [1–3] The serine modulation occurs
through rare Vmax-type effects, and may be contrasted with
the more common regulation that directly affects the binding of substrate(s) by altering Km[4]
PGDH belongs to a family of D-2-hydroxyacid dehydrogenases that includes formate dehydrogenase,
D-glycerate dehydrogenase, D-lactate dehydrogenase, ery-thronate-4-phosphate dehydrogenase, D-2-isocaproate dehydrogenase and vancomycin resistant protein [4] The family members share 22% sequence identity and 50% sequence similarity Among theD-2-hydroxyacid dehydro-genases all members are dimeric with the exception of PGDH, which forms a homotetramer Crystallographic studies of four enzymes within this family {2nac (for-mate,dehydrogenase [5]), 1gdh (D-glycerate dehydrogenase [6]), 2dld (D-lactate dehydrogenase), 1psd (3-phosphoglycer-ate dehydrogenase [7,8]} have revealed a striking similarity
in their conformations, except for the additional regulatory domain in PGDH
The crystal structure of the PGDH:NAD:serine complex [7] is depicted in Fig 1 and illustrates both the domain and quaternary arrangements The 222 symmetric tetramer has four binding sites for both serine and NADH The donut-like appearance of PGDH is similar to the tetrameric form
of glycerol kinase [9], another enzyme that is regulated by
Vmax-type kinetic changes The interface encompassing adjacent nucleotide binding domains is labeled I, and this subunit:subunit contact is shared among allD -2-hydroxy-acid dehydrogenases The additional regulatory domain forms an important new subunit interface, labeled II The two serine-binding sites located at each interface are comprised of residues from both subunits As will be shown
Correspondence to L J Banaszak, 6-155 Jackson Hall,
Department of Biochemistry, Molecular Biology and Biophysics,
University of Minnesota, 321 Church St S.E., Minneapolis,
MN 55455, USA.
Fax: + 1 612 625 2163, Tel.: + 1 612 626 6597,
E-mail: len_b@dcmir.med.umn.edu
Abbreviations: PGDH, D -3-phosphoglycerate dehydrogenase;
NSD, nucleotide and substrate domains; RBD, regulatory binding
domain; IPTG, isopropyl thio-b- D -galactoside; FDH, formate
dehy-drogenase; LDH, lactate dehydehy-drogenase; a-KG, a-ketoglutarate;
PHP, 3-phosphohydroxypyruvate; 3GriP, 3-phosphoglycerate;
DLS, dynamic light scattering; D t , translational diffusion
constant; PFG, pulsed-field gradient.
Enzymes: D -3-phosphoglycerate dehydrogenase (EC 1.1.1.95).
Note: a website can be found at http://biosci.cbs.umn.edu/BMBB/
(Received 8 May 2002, accepted 25 June 2002)
Trang 2in this report, the tetrameric PGDH belongs to a family of
dimeric homologues but the differences in quaternary
structure are not explained solely by the presence of the
regulatory domain Finally a third proposed interface across
the middle of the PGDH toroid near the region labeled III
in Fig 1 contains essentially no intersubunit contacts except
through the visible loops, residues 160–195 These
symmet-rically related loops could form relatively close hydrophobic
and charge:charge contacts, reinforcing subunit contacts
already stabilized by the interface between nucleotide
binding domains
The conformational similarity between the family
mem-bers is visible in the stereo-drawing shown in Fig 2 where a
PGDH subunit and a formate dehydrogenase subunit have
been overlaid by the method of least-squares The 44-kDa
PGDH subunit is divided as follows: nucleotide binding
domain (residues 108–292), substrate binding domain
(residues 1–102, 304–318), and regulatory or serine-binding
domain (residues 336–410) The interconnecting
polypep-tide segment, residues 103–108, 293–303, and 319–336, may
form hinge-like regions In fact, the homologous
polypep-tide segments connecting the nucleopolypep-tide- and
substrate-binding domains in the dimeric family members have been
shown to have conformational variability [5,6,10] Of equal
relevance, but not shown in Fig 2, the nucleotide binding
domains of PGDH associate into a dimer interface entirely
homologous with the quaternary structures of dimeric formate [5], glycerate [6] andD-lactate dehydrogenase Using this well-defined homology, the potential confor-mational changes associated with the inhibited vs the active form of PGDH were postulated from the crystal structures
of apo- and holo-formate dehydrogenase [5] These crystal complexes revealed that the active site cleft, formed by nucleotide and substrate binding domains, rotated 7.5 into
a more closed conformation when ligand was bound The constraints of the tetrameric nature of PGDH suggest that a similar rotation of the nucleotide and substrate domains into a more closed conformation at the active site would require additional relaxation of interactions at the regula-tory domain interface
The study of proposed domain movements were exam-ined by subcloning portions of PGDH to look at the contribution of the tetrameric structure to catalysis, stability and potential conformational changes at the serine site upon ligand binding Several chimers consisting of the nucleotide and substrate domains with variable N- and C-termini were made to resemble counterparts in the 2-hydroxyacid dehydrogenase family The kinetic properties and oligo-meric states of these truncated enzymes were determined and compared to intact PGDH In addition, the regulatory binding domain, RBD, was subcloned to create a smaller model of the serine-binding pocket that could be manipu-lated for structural study by NMR and evaluated for conformational changes upon ligand binding
M A T E R I A L S A N D M E T H O D S The expression vector, pSAWT containing the serA gene was described previously [11] The plasmids, pTrc99A and pGEX-2T, were from Pharmacia Biotech PfuDNA polymerase and the SURE cell line were from Stratagene
Fig 2 Stereoview of PGDH and the homologous formate dehydro-genase The crystallographic coordinates of formate dehydrogenase and PGDH have been superimposed by the least-squares methods The resulting overlay of the two subunits is shown in stereo with formate dehydrogenase in red and PGDH in blue A stick represen-tation NAD bound to FDH (purple) and PGDH (green) is also shown The regulatory domain of PGDH is at the top followed by the sub-strate binding domain and finally the NAD binding domain at the bottom of the figure The overlay of the two coordinate sets illustrates the close conformational homology between the two enzymes includ-ing the positioninclud-ing of the bound coenzyme.
6
Fig 1 Structure of PGDH:a summary of structure and mutations The
cartoon illustrates the crystal structure of the serine-inhibited form of
PGDH Three of the subunits of the homotetramer are colored gray.
The fourth subunit shows the three component domains,
nucleotide-binding domain (blue), the substrate-nucleotide-binding domain (red) and a
regulatory domain (green) Three arrows mark: (I) the
nucleotide-binding domain interface, and (II) the tetramer interface formed by the
interactions of two regulatory domains and (III) unobserved contact
across the middle of the tetramer The position of two of the four serine
molecules is shown by van der Waal’s surface at the regulatory
inter-face on the left Also shown in van der Waal’s surinter-faces, the NAD
molecule binds within the active site cleft along the top of the
nucleo-tide domain The numbers 1–7 on the left indicate the Ca positions of
the truncated enzymes Numbers 1–4 and number 7 describe the NSD
enzymes Specifically, numbers 1 and 2 show the position of the N
terminus, residue 7 (the first ordered residue in the crystal structure)
and 10, respectively The blue carbon atoms 3 and 4 indicate residues
314 and 317 at the C-terminus of two of the NSD proteins Residue
336, u sed in both the NSD and RBD proteins, is indicated by the green
Ca ball.
5
Trang 3The BLR cell line was from Novagen Restriction enzymes
and ligase came from either Promega or Boehringer
Mann-heim Oligonucleotide primers were synthesized by the
Microchemical Facility at the University of Minnesota, or
out-sourced via this facility DNA gel purification chemicals
were from the Bio-Rad PCR Cleanup Kit was from
Promega The Microchemical Facility at the University of
Minnesota confirmed the sequences of DNA inserts All
other chemicals were from Sigma unless otherwise noted
Mutagenesis
The nucleotide and substrate domain constructs of residues
1–336 (NSD:336) and 1–317 (NSD:317) were subcloned
from the pSAWT vector using common PCR techniques
into the pTrc99A vector The NcoI site at the 5¢ end of the
serAgene was conserved and a stop codon and unique XbaI
site were introduced at the new 3¢ terminus at residue 336 or
317 The NSD:10–314 and NSD:10–317 mutants were
constructed using the Stratagene Quik ChangeTM
mutagen-esis kit and the NSD:336:pTrc99A vector as the parental
DNA The RBD:336–410 protein, residues 336–410, was
made using the same technique as the NSD constructs, but
with a BamHI site introduced at the 5¢ end and a HindIII
site at the 3¢ end The PCR product was ligated into the
pGEX-2T vector All mutant sequences were confirmed by
DNA sequencing
Expression and purification
NSD.NSD vectors were transformed into competent SURE
cells Six 1-L flasks of 2· YT broth plus 150 lgÆmL)1
ampicillin were grown at 37C until the optical density at
600 nm reached 0.6–0.8 Protein expression was induced
with 1–1.5 mMisopropyl thio-b-D-galactoside (IPTG) After
induction cells were grown for 14 h at 22 C Cell pellets
were resuspended in 50 mM KH2PO4 pH 7.0, 2 mM
dith-iothreitol, 1 mM EDTA and 0.05% NaN3 (buffer B) and
lysed by sonication The remainder of the purification
protocol has been described previously [11] Purified protein
was concentrated using a Centriprep 10K (Amicon) and
dialyzed into buffer B Protein concentration was
deter-mined by Bradford assay and/or UV spectra using an
extinction coefficient of 0.67M )1Æcm)1 Protein was stored at
4C
RBD:336–410 RBD:336–410 plasmid was transformed
into competent BLR cells Six 1-L flasks of 2· YT broth
plus antibiotic were grown at 37C to D600¼ 0.6–1.0 and
then induced with 1 mMIPTG Cells were grown for 14 h
at 22C The cell pellet was resuspended in STE (10 mM
Tris/HCl pH 8.0, 1 mM EDTA, 150 mM NaCl) and
incubated on ice with 0.1 mgÆmL)1 lysozyme for 15 min
The solution was brought to 5 mMdithiothreitol, 2% (w/v)
sarkosyl and sonicated The mixture was stirred at 4C for
30 min followed by centrifugation at 10 000 g
Polyethyleneamine (0.035%) was added to remove DNA/
RNA, stirred at 4C for 30 min and then respun for 30 min
at 10 000 g The supernatant was concentrated using an
Amicon concentrator with a PM10 membrane (3 kDa
cut-off) and dialyzed into NaCl/Pi/EDTA (16 mM Na2HPO4,
4 mMNaH2PO4, 150 mMNaCl, 1 mMEDTA pH 7.3) The
dialyzed lysate was respun to remove particulates and
loaded onto a glutathione S-transferase (GST) affinity column (Novagen) The column was washed with 10 column vols NaCl/Pi/EDTA and then incubated with
250 U thrombin overnight at room temperature The cleaved RBD:336–410 was eluted, concentrated using a Centriprep 3K (Amicon), and stored at 4C Protein concentration was calculated from UV spectra using an extinction coefficient of 0.47M )1Æcm)1 for RBD:336–410 The identity of the protein was confirmed by N-terminal sequencing of the first 10 residues and amino acid analysis (Microchemical Facility, University of Minnesota, MN, USA)
Kinetic analysis The steady-state initial rates were determined by following either the reduction of 3-PHP or a-ketoglutarate (a-KG) The reaction was set u p with a satu rating concentration of NADH (100–200 lM) and varied concentrations of PHP (1–100 lM) or a-KG (10.4–5000 lM) at 25C The enzyme concentration for the a-KG studies was 1 lM and 0.1–0.5 lM for the PHP reactions The assay buffer for the a-KG reactions was 50 mMTris pH 7.5, 1 mMEDTA and 2 mMdithiothreitol For the 3-PHP reactions, the Tris concentration was increased to 500 mM The reaction was initiated by the addition of substrate and the decrease in
D340 monitored for 10–20 s The initial rates were deter-mined by fitting a linear regression to the curve and calculating the slope using CARY50 kinetics software Assays were repeated a minimum of five times The data were analyzed by Michaelis–Menten or Lineweaver–Burke plots and kinetic parameters derived using the SIGMA PLOT5.0 software (Jandel Scientific Inc.)
Dynamic light scattering experiments Dynamic light scattering (DLS) experiments were conducted
in Buffer B for PGDH and the NSD proteins RBD:336–410 experiments were done in NaCl/Pi/EDTA For each concentration measured, the protein was spun at
14 000 g for 10 min and passed through a 0.1-lMfilter A 12-lL sample was equilibrated by a built-in thermostat at
5C increments Data were collected with a Protein Solutions DLS system and evaluated with the DYNAPRO
V4.0 software For each temperature 15–20 data points were collected Mean values were calculated for the DLS parameters Points that were outside 1 SD were excluded Data were plotted inSIGMA PLOT5.0
Gel filtration experiments Gel filtration experiments were performed in Buffer B PGDH (2 mgÆmL)1), NSD:317 (2 mgÆmL)1), or
dehydrogenase (D-LDH) (2 mgÆmL)1) were ru n over a Sephacryl S200 (Pharmacia) gel filtration column with both low molecular weight standards (ribonuclease A, 13.7 kDa; chymotrypsinogen A, 25 kDa; ovalbumin,
43 kDa; BSA, 67) (Run 1) and high molecular weight standards (aldolase, 158 kDa; catalase, 232 kDa; ferritin,
440 kDa; thyroglobulin, 669 kDa) (Run 2) Chromato-graph profiles were calculated from the absorbance of the fractions at 280 nm for the molecular mass standards and activity measurements for PGDH, NSD:317 and -LDH
Trang 4The molecular weights of PGDH, NSD:317 andD-LDH
were calculated from the linear regression of Kav[(Ve) Vo)/
(Vt) Vo), where Veis the elution volume, Vois the void
volume and Vt is the total volume] vs the log of the
molecular weight of the standards
CD
RBD:336–410 experiments were performed in NaCl/Pi/
EDTA CD spectra were collected on protein (0.722ÆmgÆ
mL)1) in the presence or absence of 1 mMserine A buffer
blank was completed for both the buffer and buffer plus
1 mM serine The spectra were collected on a Jasco 710
instrument at room temperature using a 0.05-mm quartz
cell Spectra were collected from 250 to 200 nm with eight
accumulations The data were averaged over the
accumu-lations, corrected for the buffer blank and random signals
were smoothed using the JASCO software package Data
were exported toSIGMA PLOT5.0 for analysis
Pulsed-field gradient NMR
To corroborate the DLS measurements, pulsed-field
gradi-ent (PFG)-NMR [12,13] was used to give an independgradi-ent
determination of the translational diffusion constant (Dt)
for the protein RBD:336–410 NMR was carried out in
collaboration with the Mayo laboratory at the University of
Minnesota Spectra were collected and analyzed by Shou
Lin Chang of the Mayo laboratory In the PFG-NMR
experiment, Bo, constant magnetic field, was superimposed
twice during a short time interval, d, by an additional
inhomogenous gradient (Gz) The result of the two gradient
pulses is to create an echo If no motion or relaxation
occurred on the z-axis, the echo would have been identical
to the initial signal However, the observed echo will be
attenuated by both relaxation and random motion
(diffu-sion), along the z-axis The attenuation, A(t) can be
described by:
AðtÞ ¼ Að0ÞexpðRðtÞ c2G2Dtd2ðd d=3ÞÞ
where R(t) is attenuation due to relaxation, c is the
magnetogyric ratio, G is the gradient strength, d is the
duration of the gradient pulse, and D is the interval between
the start of the two gradient pulses To determine an
accurate measurement of Dt, a series of 12 one-dimensional
PFG spectra were collected at gradient field strengths, 0, 5,
10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 GÆcm)1 The data
were then fit to the semi-log of the equation above to
determine the value of Dt Experiments were conducted at
both 10 and 25C Protein (1 mM) was in 50 mM
KH2PO4, pH 6.5, in D2O Serine, when present, was at
1 mM The experiments were carried out on a Varian
UNITY 600 MHz NMR with triple resonance probe and
triple axis gradient unit (High Magnetic Field Facility,
University of Minnesota)
R E S U L T S A N D D I S C U S S I O N
Nucleotide substrate somains from GriP DH
The preparation of a monodisperse form of PGDH
insensitive to the presence of serine but fully active was
designed based on a previously determined crystal structure (Fig 1) [7] Removal of the serine-binding domain was predicted to eliminate allosteric inhibition by serine and produce a dimeric enzyme As shown in the data below, manipulation of PGDHs quaternary structure was far more complicated and it was not possible to obtain a dimeric enzyme Several variations of the NSD’s were developed using the standard PCR technique of introducing a stop codon and unique restriction site at the desired termination point
NSD:336 (residues 1–336), the first two-domain protein
to be made, was soluble and yielded 5–12 mg per 6 L ferment However, NSD:336 included a segment of the linker sequence between the substrate and the regulatory domains, and tended to form higher oligomeric species (data not shown) This extended linker may have decreased stability and provide a site of aggregation, and was therefore removed in another form NSD:317 (residues 1–317) NSD:317 was monodisperse in solution and relatively stable (see below), and therefore more amenable to study Two other two-domain enzymes were also created: NSD:10–314 and NSD:10–317 These forms eliminated the N-terminal segment that was disordered in crystalline PGDH with serine The recombinant products were largely insoluble and further studies were aban-doned
Kinetic evaluation of NSDs
As conformational changes had been linked to the catalytic activity of both PGDH and formate dehydrogenase (FDH) [5,14] removal of the regulatory domain was hypothesized
to have an effect on the kinetic parameters of PGDH The steady-state parameters are reported for two of the chimers although our primary focus was NSD:317 because the quaternary structure of this enzyme was definable The activities of PGDH and NSD:317 were assayed following the reduction of PHP, which occurs 70-fold faster than the oxidation of GriP [15], or the alternate substrate, a-KG [16] Although PHP and a-KG are three- and five-carbon substrates, respectively, the fourth carbon and 5-carboxyl
of a-KG are similar to the bulky phosphate group in PHP The results of the steady-state kinetic studies are summa-rized in Table 1 For both the PHP and a-KG assays, substrate inhibition was observed at high concentrations (Fig 3), possibly due to the slow release of oxidized cofactor and leading to an abortive complex of substrate/ NAD The data from the reduction of PHP and a-KG, excluding data exhibiting substrate inhibition, were evalu-ated by Michaelis–Menten plots to derive Kmand Vmax Overall the kinetic parameters of the native and NSD enzymes do not vary significantly (Table 1) The Km(PHP) for PGDH agrees well with the value first published
by Pizer, 1.2 ± 1 vs 1.3 lM[15] The alternative substrate, a-KG, shows an 18-fold increase in Kmover PHP and an order of magnitude decrease in Vmax/Km The lower catalytic efficiency is consistent with the hypothesis that the 5-carboxyl group in a-KG is not a good substitute for the phosphate group of PHP However, both NSD:317 and PGDH behave similarly with respect to this pseudo-substrate
The effect of serine on NSD:317 was also tested Using saturating concentrations of both substrate and cofactor in
Trang 5the presence and absence of 5 mM serine (IC50for native
enzyme¼ 5 lM; [17]), no change in the initial rate of the
catalytic reaction was found (data not shown) Given that
the NSD enzymes were not affected by serine, the purity of
an enzyme preparation, usually contaminated with
wild-type PGDH from Escherichia coli, was routinely determined
by assays in the presence and absence of serine Because the
kinetic characteristics of NSD:317 are comparable to those
of the native enzyme, the release of the hinged active site
from the constraints of the regulatory domain have neither
increased nor decreased its catalytic capabilities This
reinforces the supposition that the serine-binding domain
evolved solely for regulation, and may explain also why the
mammalian forms of the enzyme, although no longer
regulated by serine [18], have not shed the serine-binding
domain
Quaternary structure and stability
As shown in Fig 1, the PGDH tetramer has two major types of subunit interfaces Removal of the subunit contacts formed by the regulatory domains, as in the NSD enzymes, was predicted to result in a dimeric species DLS results from solutions of NSD:336 at micromolar subunit concen-trations indicated that this enzyme formed higher oligo-meric species, up to 12-mers (data not shown) The removal
of the C-terminal linker region (residues 318–336) in the NSD:317 enzyme alleviated the aggregation problem
An overview of the Dt for NSD:317 compared to the native enzyme and the concentration dependence is shown in Fig 4 In contrast to NSD:336 protein, this truncated form gave reproducible measurements at 0.5, 1.0 and 2.0 mgÆmL)1(14.7–58.7 lM) The Dtvalues are slightly larger than those for the native enzyme up to 30C, consistent with NSD:317 forming a somewhat smaller molecule
The Dtdata were analyzed by two different methods, both of which are summarized in the insert to Fig 4 Using the Stokes–Einstein equation, Dtmay be u sed to calcu late the equivalent hydrodynamic radius, Rh:
Dt¼ kT=6pgRh where k is the Boltzman constant, T is the absolute temperature and g is the solvent viscosity As shown in the inset, Dts of 440 and 520 for PGDH and NSD:317, respectively, lead to Rh values of 52 A˚ and 47 A˚ The corresponding molecular weights of PGDH and NSD:317, based upon a spherical model, were 157 and 126 kDa respectively Given that the subunit molecular mass (m) of NSD:317 is 34 kDa, these results suggested that the truncated enzyme was forming a tetramer instead of the expected dimer
The second method of evaluating Dtmakes use of the crystallographic model coordinates of PGDH If the coordinates are used to determine a prolate ellipsoid of equivalent dimensions, Rh, of a comparable sphere may be calculated:
Rh¼ ðab2Þ1=3 where a and b are the half lengths of the long and short axis
of the crystallographic prolate ellipsoid, respectively The proposed structure of NSD as either a dimer, as expected, or
Table 1 Steady state properties of NSD:336, NSD:317 and D -3-phosphoglycerate dehydrogenase Rates of NADH oxidation were determined by measuring the decrease in OD at 340 nm The a-KG assays were completed in 50 m M Tris, pH 8.0, 2 m M dithiothreitol, 1 m M EDTA with saturating cofactor, 200 l M , and a-KG concentrations from 10.4 to 5000 l M at 25 C The 3-phosphohydroxypyruvate assays were carried out with a 10-fold higher concentration of Tris, 500 m M , and 3-phosphohydroxypyruvate concentrations from 1 to 100 l M
K m a
l M
V max a
s)1
V max /K m a
s)1Æ M )1
a Parameters derived from fitting the velocity vs substrate concentration plot to the Michaelis–Menten equation.
Fig 3 Michaelis–Menten plot of PGDH, NSD:317 and NSD:336
ki-netic data for the a-KG substrate The velocity vs substrate
concen-tration plots of the kinetic data for PGDH (d), NSD:317 (s)
and NSD:336 (m) clearly show that no significant differences between
kinetic parameters are distinguishable The largest difference occurs in
the value of V max but this is less than a twofold difference between
native and truncated enzymes At a-KG concentrations > 2–3 m M ,
substrate inhibition was observed Data points exhibiting inhibition
(shaded in grey) were excluded from calculation of the kinetic
parameters Experimental conditions are given in Table 1 Similar data
were collected with PHP as the substrate, not shown The y-axis, v, is
defined as [NADH]/[enzyme] with units of s)1.
Trang 6a tetramer, utilizing contacts of the extended loops across
the ellipsoid, were modeled from the PGDH coordinates by
removing the regulatory domain The inset of Fig 4
summarizes the results of these approximations The
agreement between the observed Rhand the Rhcalculated
from the crystallographic ellipsoid is consistent with a
tetrameric form of NSD:317
The unexpected results of the DLS experiments
suggest-ing a tetrameric form of the NSD:317 enzyme was
confirmed by gel filtration The chromatographs of PGDH
(predicted m 176 kDa) and NSD:317 (predicted dimeric m
68 kDa, predicted tetrameric m 136 kDa) revealed that
both enzymes were eluting before the molecular mass
standard aldolase (m 158 kDa) (Fig 5) In fact, PGDH
coeluted with the molecular mass standard catalase (m
232 kDa) at a higher than predicted molecular mass,
indicating that the ellipsoidal quaternary structure has
affected its elution pattern To evaluate the oligomeric state
of NSD:317 while allowing for the overall shape of the
molecule, we compared its elution pattern with that of a
known dimericD-2-hydroxyacid dehydrogenase of similar
fold,D-LDH (predicted dimeric m 74 kDa) [19] (1ldh)
The D-LDH elution profile indicates that this enzyme
forms both a dimer (majority) and a tetramer [19], with
predicted molecular masses of 74 and 148 kDa, respectively
NSD:317 elutes slightly after the tetrameric formD-LDH
but significantly before the dimeric form ofD-LDH The
differences in tetrameric molecular mass of D-LDH and
NSD:317 may result from D-LDH being slightly larger
(subunit m of 37 kDa vs 34 kDa) or reflect a tighter
packing of the tetramer form of NSD:317 leading to a more compact and thus smaller species If the elution profiles of the well characterized PGDH, the tetramericD-LDH and dimeric D-LDH are used to determine a molecular mass standard curve, the mass of NSD:317 would be calculated
as 141.8 kDa compared to the predicted tetrameric mass of
136 kDa Therefore, gel filtration results of nonspherical proteins greatly benefit from evaluation with respect to proteins of known similar folds and quaternary structure The results of the gel filtration studies are consistent with the DLS data in support of a tetrameric form for NSD:317 The DLS measurements were also used to evaluate the stability of NSD:317 in comparison to PGDH by monitor-ing Dtas a function of temperature The Dtvalues for the NSD:317 dropped dramatically above 30C compared to native enzyme, indicative of formation of a larger species In addition, the polydispersity, that was negligible below
30C, rises considerably The decreased stability of NSD:317 and the length dependence of the C-terminus to determine monodispersity are consistent with the now exposed substrate:regulatory domain contact potentially offering a site of aggregation or preliminary unfolding As mentioned above, mammalian PGDH retains its regulatory binding domain although it no longer allosterically
regulat-ed by serine Perhaps, the RBD has been retainregulat-ed to increase protein stability and limit aggregation
Fig 4 DLS of NSD:317 The DLS experiments were conducted as a
function of both temperature and concentration D t , increases, as
predicted by the Stokes–Einstein equation, with temperature to 30 C.
At 35 C the D t value decreases by approximately one-third,
sug-gesting that the protein has begun to aggregate Native enzyme
is shown as closed circles, mutant as open symbols The increase in
D t for NSD:317 does not appear to be concentration dependent
over this concentration range, 0.5 mgÆmL)1(s), 1 mgÆmL)1(h) and
2 mgÆmL)1 (n) The inset compares the calculation R h , from the
experimental D t and the Stokes–Einstein equation vs calculation from
the crystallographic structure and a prolate ellipsoid The values of
a and b are the length of the two axes of the ellipsoid measured from
the crystal structure, 1psd DLS measurements were conducted in
50 m M KH 2 PO 4 pH 7.0, 2 m M dithiothreitol, 1 m M EDTA, 0.05%
NaN 3 At a given temperature the values for each parameter were
averaged for the 0.5, 1.0 and 2.0 mgÆmL)1measurements.
Fig 5 Gel filtration chromatograph of PGDH and NSD:317 The elution profiles of PGDH (m, 176 kDa; d), NSD:317 (j), and D -LDH (m, 74 kDa dimeric; m, 148 kDa tetrameric; m) are shown with respect
to the profile of molecular weight standards, catalase (m, 232 kDa), aldolase (m, 158 kDa) and ovalbumin (m, 43 kDa) depicted by the gray line PGDH elutes with catalase suggesting that the ellipsoidal shape of the enzyme increases the apparent molecular mass D -LDH appears to run as a dimer, D -LDH 1, and tetramer, D -LDH 2, with the majority seen as a dimer Both D -LDH species elute at a higher than predicted molecular mass (100 kDa and 220 kDa), again this observed increase in molecular mass can be attributed to the elongated shape of the enzyme The comparison of the NSD:317 elution with the D -LDH pattern suggests that the truncated enzyme is forming a tetramer with a molecular mass of 196 kDa (predicted m, 136 kDa) Gel filtration studies were completed in Buffer B on a Sephacryl S200 matrix with each protein sample at a concentration of 2 mgÆmL)1 Note that the elution of PGDH, NSD:317 and D -LDH were determined by activity measurements to remove ambiguity of elution profiles from absor-bance measurements at 280 nm.
Trang 7Regulatory domain
The regulatory or serine-binding domain of PGDH consists
of 76 residues (residues 336–410) In the crystal structure,
the subunit–subunit interface at the regulatory domains (II
in Fig 1) was shown to consist of an extended b sheet
created by adjacent subunits [7] Serine binding was
proposed to increase the interactions at this interface
thereby locking the active site into a more open and inactive
conformation The uninhibited form of the enzyme would
be more flexible at the interface formed by the regulatory
domains allowing more motion at the hinge regions and
permitting the active site to close To allow this
conforma-tional flexibility, changes at the interface formed by the
regulatory domains were proposed to involve the disruption
of the extended b sheet
To study the effect of serine binding at this subunit
interface, we attempted to develop a simple dimer of the
regulatory domains The small size of this domain, 76 residues, would allow for structural studies by NMR or crystallography However, polypeptides of this molecular weight proved difficult to purify from E coli extracts, so RBD:336–410 was expressed as a GST fusion protein After cell lysis, SDS gels indicated that the majority of the target protein was in the resulting insoluble pellet Addition of a detergent, sarkosyl, solubilized much of the GST-RBD:336–410 RBD:336–410 could be obtained in pure form by chromatography on a glutathione column followed
by proteolysis with thrombin to remove the GST tag (data not shown)
Unlike the NSD proteins, RBD:336–410 could not be characterized by a catalytic assay The chemical identity of this small, purified protein was verified by both amino acid analysis and N-terminal sequencing of the first 10 residues
As the protein was solubilized with detergent, CD measurements were conducted to determine whether stable secondary structure had formed The CD measurements were completed in the presence and absence of serine Fig 6 shows that RBD:336–410 had minima for both
b structure (217 nm) and a helix (222 and 208 nm) The addition of serine had no significant effect on the secondary structure The CD spectra show the presence
of secondary structural elements consistent with the intact enzyme
To determine the oligomeric nature of RBD:336–410, both DLS experiments at 18 and 23C (0.5 mgÆmL)1) and PFG-NMR studies in collaboration with the Mayo laboratory at the University of Minnesota were carried out If RBD:336–410 was dimeric, this would be apparent in the Dt and the corresponding Rh The NMR studies would also be useful for determining whether the structure of RBD:336–410 could be solved
by NMR The results of these experiments, shown in Table 2, indicated that the new protein formed not the expected dimeric species, but a higher oligomeric mole-cule
In Table 2, values of Rhare based on the Stokes–Einstein relationship mentioned earlier The results from the two experimentally independent methods, NMR and DLS, agree within 10% Furthermore, the data in Table 2 indicate that the addition of serine had no significant effect on the Dt values Given the consistency of the data, taking the overall average appeared justified resulting in an Rhof 37 A˚ Using
a partial specific volume of 0.73 mLÆg)1, the molecular mass
of the new aggregate would be 42 kDa With a monomeric molecular mass for RBD:336–410 of 8.1 kDa, the regula-tory domain by itself behaves like either a pentamer or a
Fig 6 CD spectra of RBD:336–410 in the presence/absence of 1 m M
serine CD was performed in 16 m M Na 2 HPO 4 , 4 m M NaH 2 PO 4 ,
150 m M NaCl, 1 m M EDTA, pH 7.3 at 0.72 mgÆmL)1( 0.1 m M )
protein Serine, when present, was at 1 m M The RBD:336–410 spectra
are shown as black lines: RBD:336–410 + 1 m M Serine are shown as
gray lines RBD:336–410 contains two minima at 217–222 and 206–
208 nm corresponding to a-helical and b strand content, respectively.
The addition of serine to RBD:336–410 does not have a significant
effect on the secondary structure.
Table 2 DT for RBD:336–410 calculated from DLS and PFG-NMR data DLS experiments were conducted in 16 m M Na 2 HPO 4 , 4 m M NaH 2 PO 4 ,
150 m M NaCl, 1 m M EDTA pH 7.3 in the presence and absence of 1 m M serine as indicated NMR studies were completed on protein at 1 m M under identical conditions D t s are reported in cm2Æs)1· 109 R h (equivalent sphere) was calculated using the Stokes–Einstein model.
Trang 8hexamer Because of the inherent shape uncertainties in
extrapolating molecular masses from DT, and in spite of the
close agreement between the two independent methods, the
exact oligomeric state and nature of subunit interactions
remains unresolved The results, however, do clearly
indicate that the RBD does not form the expected dimer
C O N C L U S I O N S
The NSD enzymes were developed as an alternative to the
serine regulated native PGDH Removal of the regulatory
domain had little influence on the enzyme’s catalytic
reaction and kinetic parameters determined from
steady-state studies The largest differences between the native
enzyme and the NSD proteins occurred in the stability of
the enzymes The native tetramer retained its predicted DT
u p to 40C whereas the NSD protein began to aggregate
and/or denature between 30 and 35C These results are not
surprising as removal of the regulatory or serine-binding
domain exposes a surface area that is partially buried in the
native tetramer and may be susceptible to aggregation or
unfolding In mammals, serine feedback regulation has been
replaced by transcriptional control [18] yet the alignment of
sequences from a variety of species indicate that the
regulatory domain has been conserved The retention of
the regulatory domain and thus the subunit:subunit
inter-face may provide additional stability to the quaternary
structure as is observed in the differences between E coli
PGDH and the NSD enzymes Mutations within this
domain of the human PGDH lead to loss of or lowered
serine production without a significant decrease in mRNA
production [20] The work presented here would suggest
that stability studies of these clinically characterized
muta-tions may give insight as to the role of the regulatory
domain in higher eukaryotes
We predicted that the NSDs would more closely resemble
other dimericD-2-hydroxyacid dehydrogenases The
oligo-meric structure of NSD:317 was, instead, a tetramer From
the crystallographic structure of the serine-inhibited
en-zyme, and some preliminary structural results with a mutant
form of PGDH, a model has been formulated Figure 7A,B
reiterate the subunit contacts of the PGDH–NAD–serine
structure and the proposed conformational change upon
catalysis or release of inhibition Given that the tetrameric
interface, labeled II in Fig 7, had been removed, NSD:317
must have formed a new subunit–subunit interface to
remain a tetramer New structural results from a point
mutation, W139G PGDH, have shown the collapse of the
ellipsoid with extensive interactions being made between the
extended loops (residues 165–190) and the subunits across
the toroid [21] Based upon this new structural data we
propose that the NSD:317 enzyme has formed a new, or as
yet structurally uncharacterized, tetrameric interface
through the interaction of the extended loops (residues
165–190) (Fig 7C) Perhaps similar subunit:subunit
inter-actions are important in the uninhibited form of PGDH, in
which the active site cleft has adopted a closed
conforma-tion Structural studies to investigate that possibility are
currently underway
The subcloning of the regulatory binding domain offered
a unique opportunity to look at conformational changes
induced by serine as a subset of the whole enzyme
However, the construct proved poorly soluble unless it
was coupled with a fusion protein and solubilized with detergents (sarkosyl) The presence of the secondary struc-ture as assessed by CD spectra suggested that the regulatory domain could fold independently However, DLS and PFG-NMR experiments clearly showed that the protein aggregated under a variety of conditions The aggregation tendency coupled with the small size makes this domain particularly difficult to analyze with respect to ligand binding Nonetheless, the DT values obtained from solu-tions of RBD were nearly identical whether determined by DLS or NMR This establishes the usefulness of both methods in studying the hydrodynamic properties and quaternary structures of macromolecules, and
demonstrat-ed that the regulatory domains alone form an even more complex quaternary structure
The new enzymes created by recombinant methods provided a step back in the evolutionary chain Rather than stringing together multiple functional units we can dissect the contribution of individual domains towards the complex regulation and cooperativity observed within this enzyme system The role of the tetrameric PGDH evolved to provide a means of regulating serine production within prokaryotes and lower plants Although at the outset we predicted, based upon PGDH structural data and homol-ogous dimeric enzymes, an easily manipulated oligomeric structure, we were foiled by the complexities of heretofore unrevealed subunit:subunit contacts Loss of one of the obvious tetrameric interfaces still results in a tetrameric enzyme We continue our studies of this new subunit contact by looking at the native enzyme and why this interface may be beneficial
Fig 7 Model of regulatory domain subunit:subunit interface proposed conformational changes In this representation of PGDH only half of the tetramer is depicted The domains are labeled NAD-BD, nucleo-tide binding domain; SBD, substrate binding domain; and RBD, regulatory binding domain The arrows describe the positions of twofold rotation axes in the plane of the drawing The third dyad associated with the 222 symmetrical tetramer is indicated by the black ellipse located at the intersection of the dyad arrows In the inhibited state of PGDH (A), serine molecules are depicted as black stars, and the regulatory domains form an extended b sheet with the serine molecules bridging the two subunits The crosses (substrate) located between the SBDs and NAD-BD domains indicate the location of the active sites In this schematic model, the uninhibited state of PGDH (B) differs by the reorientation of all three domains The new confor-mational state now contains a more closed conformation at the active site The NSDs in (C) lack the RBDs In this form, new subunit interfaces form across the dyad perpendicular to the plane of the drawing and a tetramer resu lts.
Trang 9A C K N O W L E D G E M E N T S
This work was funded by National Science Foundation grants
MCB9318699 to L J B and MCB9986278 to J E B and a grant
from the National Institutes of Health (GM56676) to G A G The
authors are grateful to both M Lees and J Bratt of the Banaszak
laboratory for assistance in preparation of DNA constructs and protein
purification The authors would also like to thank Shou Lin Chang of
the Mayo laboratory at the University of Minnesota for conducting the
PFG-NMR experiments and K Mayo for use of the Jasco 710 CD
spectrophotometer We gratefully acknowledge the help of J Barycki
in the preparation of this report.
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