Crystal structure of human senescence marker protein 30: Insights linking structural, enzymatic, and physiological functions

Article abstract of DOI:10.1021/bi9022297

Human senescence marker protein 30 (SMP30), which functions enzymatically as a lactonase, hydrolyzes various carbohydrate lactones. The penultimate step in vitamin-C biosynthesis is catalyzed by this enzyme in nonprimate mammals. It has also been implicated as an organophosphate hydrolase, with the ability to hydrolyze diisopropyl phosphofluoridate and other nerve agents. SMP30 was originally identified as an aging marker protein, whose expression decreased androgen independently in aging cells. SMP30 is also referred to as regucalcin and has been suggested to have functions in calcium homeostasis. The crystal structure of the human enzyme has been solved from X-ray diffraction data collected to a resolution of 1.4 .... The protein has a 6-bladed I-propeller fold, and it contains a single metal ion. Crystal structures have been solved with the metal site bound with either a Ca²⁺ or a Zn ²⁺ atom. The catalytic role of the metal ion has been confirmed by mutagenesis of the metal coordinating residues. Kinetic studies using the substrate gluconolactone showed a k_cat preference of divalent cations in the order Zn²⁺ > Mn²⁺ > Ca²⁺ > Mg²⁺. Notably, the Ca²⁺ had a significantly higher value of K_d compared to those of the other metal ions tested (566, 82, 7, and 0.6 μm for Ca²⁺, Mg²⁺, Zn²⁺, and Mn ²⁺, respectively), suggesting that the Ca²⁺-bound form may be physiologically relevant for stressed cells with an elevated free calcium level.

Full text of DOI:10.1021/bi9022297

3
436 Biochemistry 2010, 49, 3436–3444
DOI: 10.1021/bi9022297
Crystal Structure of Human Senescence Marker Protein 30: Insights Linking Structural,
†,‡
Enzymatic, and Physiological Functions
§
Subhendu Chakraborti and Brian J. Bahnson*
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716.
Present address: University of California San Francisco, Department of Anesthesia and Perioperative Care.
§
Received December 31, 2009; Revised Manuscript Received March 2, 2010
ABSTRACT: Human senescence marker protein 30 (SMP30), which functions enzymatically as a lactonase,
hydrolyzes various carbohydrate lactones. The penultimate step in vitamin-C biosynthesis is catalyzed by this
enzyme in nonprimate mammals. It has also been implicated as an organophosphate hydrolase, with the
ability to hydrolyze diisopropyl phosphofluoridate and other nerve agents. SMP30 was originally identified as
an aging marker protein, whose expression decreased androgen independently in aging cells. SMP30 is also
referred to as regucalcin and has been suggested to have functions in calcium homeostasis. The crystal
˚
structure of the human enzyme has been solved from X-ray diffraction data collected to a resolution of 1.4 A.
The protein has a 6-bladed β-propeller fold, and it contains a single metal ion. Crystal structures have been
2
þ
2þ
solved with the metal site bound with either a Ca or a Zn atom. The catalytic role of the metal ion has been
confirmed by mutagenesis of the metal coordinating residues. Kinetic studies using the substrate glucono-
2
þ
2þ
2þ
2þ
lactone showed a k preference of divalent cations in the order Zn > Mn > Ca > Mg . Notably, the
cat
2
þ
Ca had a significantly higher value of K compared to those of the other metal ions tested (566, 82, 7, and
.6 μm for Ca , Mg , Zn , and Mn , respectively), suggesting that the Ca -bound form may be
d
2þ
2
þ
2þ
2þ
2þ
0
physiologically relevant for stressed cells with an elevated free calcium level.
1
Senescence marker protein 30 (SMP30 ) was originally identi-
fied as an aging marker protein, whose expression decreases in an
androgen independent manner in rat liver and kidney cells (1).
Several reports have attempted to establish SMP30 as an anti-
aging factor (2-11). SMP30 is also known as regucalcin and was
reported in 1988 (12) as a calcium binding protein that does not
have an EF-hand calcium binding motif. Since then, studies have
shown that regucalcin plays a pivotal role in Ca regulation and
homeostasis in mammalian cells (13, 14).
The physiological role of SMP30 has been linked to various
enzymatic, as well as nonenzymatic functions, including its zinc
dependent gluconolactonase activity. In the penultimate step of
Although, our primary interest lies in understanding the
enzymatic function of SMP30 and its implication to human
physiology, we are also exploring its potential as an organopho-
sphorus (OP) hydrolase. In 1999, Billecke et al. (17) purified a
soluble enzyme from mouse liver that is capable of hydrolyzing
the OP compound diisopropylfluorophosphate (DFP), which
turned out to be SMP30. Subsequently, it was reported that
2þ
2þ
SMP30 could hydrolyze DFP in the presence of Mg (18). These
aforementioned reports as well as distant sequence similarities
(10% identity and 16% similarity) and a prediction of being a
6-bladed β-propeller, led us to believe that SMP30 is related to
two eukaryotic calcium dependent OP hydrolases: human serum
paraoxonase-1 (PON1) and squid diisopropylfluorophosphatase
(DFPase). PON1, which catalyzes the hydrolysis of various OP
nerve agents, such as sarin and soman, is now thought to function
as a lactonase (19). DFPase, which is structurally homologous to
PON1, was named for its ability to catalyze the hydrolysis of
DFP-like substrates (20).
vitamin-C biosynthesis in nonprimate mammals,
L-gulonate is
converted to -gulonolactone by SMP30, which functions as a
L
gulonolactonase (15). Primates are unable to synthesize their own
vitamin-C due to the absence of gulonolactone oxidase, which is
the enzyme that catalyzes the final step oxidation of gulonolac-
tone to ascorbate. This function in vitamin-C biosynthesis in
nonprimates was confirmed in a study that showed that SMP30
deficiency caused scurvy in knockout mice (16).
As there are no reported crystal structures of a eukaryotic
SMP30, our initial goal was to solve the crystal structure of
human SMP30 and to obtain an overall architecture of the
protein and its active site. We also wanted to see how the active
sites of PON1 and DFPase correspond to the SMP30 structure so
that we could have a better idea of relating these enzyme families.
Second, we wanted to understand and establish the role of metal
ions in the structure and function of SMP30. Previous studies,
†
This work was supported by NIH Grants 2P20RR015588 from the
National Center for Research Resources and 1R01HL084366 from the
National Heart, Lung, and Blood Institute.
‡
Protein Data Bank entry codes 3g4e and 3g4h are the atomic
2
þ
2þ
coordinates and structure factors for the Ca bound and the Zn
bound crystal structures of human SMP30, respectively.
*To whom correspondence should be addressed. Tel: (302) 831-0786.
2þ
with rat and mouse SMP30, have reported that Zn is the metal
Fax: (302) 831-6335. E-mail: bahnson@udel.edu.
1
2þ
Abbreviations: APS, Advanced Photon Source; DFPase, diisopro-
of choice in gluconolactonase activity (16), Mg in DFPase
pylfluorophosphatase; PON1, paraoxonase 1; rmsd, root mean squared
deviation; SMP30, senescence marker protein 30; OP, organopho-
2þ
activity (18), and Ca in cell regulation and homeostasis (14). In
contrast to the calcium-dependent PON1 and DFPase enzymes,
sphorus; PDB, protein data bank; K
d
, dissociation constant; DFP,
2þ
diisopropylphosphofluoridate.
the catalytic role of Ca in SMP30 has been brought into doubt.
pubs.acs.org/Biochemistry
Published on Web 03/23/2010
r 2010 American Chemical Society

Article
Biochemistry, Vol. 49, No. 16, 2010 3437
45
2þ
Data from a Ca -binding assay had suggested that rat SMP30
is not a calcium binding protein (18). Also, it was suggested that
mouse SMP30 does not have a calcium dependent gluconolacto-
nase activity (16).
Here, we report the first crystal structures of the human
2
þ
2þ
.
enzyme SMP30, which are bound with either Ca or Zn
Additionally, kinetic characterization of SMP30 using the sub-
2þ
strate gluconolactone demonstrates a preference for Zn with a
FIGURE 1: Reaction of gluconolactone to gluconic acid is catalyzed
by the enzyme SMP30. This reaction requires a divalent cation in the
2þ
2þ
2þ
decreasing catalytic efficiency with the Mn , Mg , and Ca
2
þ
2þ
2þ
2þ
bound forms. An overall comparison of the homologous en-
zymes, particularly PON1 and DFPase, delineates a unique role
of SMP30’s metal binding site and active site. Also, the compar-
ison of the dissociation constants of the metal ions with their free
cellular concentrations allow us to speculate that SMP30 may
switch to a calcium dependent lactonase under stressed condi-
tions that yield an increased concentration of calcium.
SMP30 active site such as Zn , Ca , Mg , or Mn .
to overnight proteolysis by thrombin (Enzyme Research
Laboratories). The proteolyzed sample was incubated with the
glutathione sepharose beads for 30 min, and then the supernatant
above the settled resin was dialyzed with a low ionic strength
buffer (25 mM Tris-HCl at pH 7.5, 1 mM CaCl2, and 1 mM
DTT) for 3 h. The sample was then passed through a 5 mL
benzamidine column (GE Healthcare) to remove thrombin, and
the flow through was concentrated to ∼5 mg/mL. The enzyme
activity was confirmed by a gluconolactonase assay (described
below) and the purity was checked by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE).
Alanine scanning mutagenesis of the metal binding residues
was performed using the Quikchange protocol (Stratagene) to
make the site-directed mutations E18A, N103A, N154A, and
D204A. The procedures used for the expression and purification
of the mutants were similar to that of the wild type enzyme.
SMP30 Enzyme Kinetics. Enzyme activity was measured
MATERIALS AND METHODS
Expression of Human SMP30. The full-length human
SMP30 cDNA (gi-22025639) was amplified by PCR using the
pRc/CMV plasmid (kindly supplied by Scott Billecke) as the
0
template. The forward and reverse primers were 5 -GCGC-
0
0
GAATTCTTCCATTAAGATTGAG-3 and 5 -GCGTCGACT-
CATCATCCCGCATA-3 , which contain the restriction sites for
0
EcoRI and SalI, respectively (in bold). The PCR-amplified DNA
fragment was ligated into the pGEX-4T-3 vector (GE
Healthcare), which has a GST tag at the N-terminus to the
full-length human SMP30 gene with an engineered thrombin-
cleavage site. The cloned human SMP30 gene was verified by
DNA sequencing.
using the substrate -glucono-δ-lactone by following the decrease
in absorbance of a pH indicator p-nitrophenol (pKa 7.1) caused
by the net stoichiometric formation of an H (Figure 1). The pH
D
þ
The SMP30-pGEX-4T-3 construct was transformed into E.
coli BL21 (DE3) RIL codon plus cells and expressed, in a
modified LB media. For protein expression and purification,
all chemicals used were obtained from Sigma-Aldrich unless
otherwise noted. One liter of Miller LB media (Fisher Sci.) was
supplemented by 10 mL of 40% glycerol (filter sterilized), 1 mL of
(6.4) of the reaction mixture was kept below the pKa (7.1) so that
the majority of the indicator species would be phenolate ion
(yellow) before the start of the reaction and any change in color
(absorbance decrease at 405 nm) would be attributed to the
stoichiometric production of H due to the enzymatic catalysis.
The reaction mixture contained 10 mM PIPES buffer (pH 6.4),
þ
0.2 M potassium phosphate buffer (pH 7.1), and 1 mM CaCl2,
10 mM
D-glucono-δ-lactone, 0.25 mM p-nitrophenol, 75 μM
and 1 M NaOH was added to bring the final pH to 7.1. Before
inoculation, 100 mg/L ampicillin and 34 mg/L chloramphenicol
were added to the sterilized media. Ten milliliter of overnight
culture was added per liter of media and grown at 37 °C until the
ZnCl , and an aliquot of enzyme in a total volume of 1 mL. The
2
decrease of absorbance at 405 nm was correlated to a change of
þ
[H ] by a standard curve with known amounts of HCl (fresh
dilutions from commercial stock). Initial velocities were repeated
at a range of substrate concentrations. Kinetic parameters of
OD
reached 2.0 at ∼4 h. The cultures were then subjected to
600 nm
þ
a cold shock and kept at 4 °C for 45 min. They were then induced
with 1 mM IPTG and grown at room temperature (25 °C) for
Vmax and KM were correlated to H production by a nonlinear fit
to v = Vmax[S]/(KM þ [S]) using the program GraphPad Prism
0
1
2-14 h. Cells were harvested by centrifugation at 5,000g and
3.0. The enzyme concentration used in assays was determined
using the BCA protein assay (Thermo Scientific) and used in the
calculation of kcat values. The experiments were repeated with
metal ion concentrations up to 1 mM and the kinetic parameters
were calculated. Standard errors for kcat and KM were determined
based on the least-squares fit of the Michaelis-Menten equation.
Dissociation Constants of Divalent Cations with
SMP30. Metal free enzyme was prepared to study the effects
of various divalent cations on gluconolactonase activity. Acid
rinsed plastic containers were used for all of the purification steps,
deionized water was passed through a chelex column (Biorad),
and the buffers were dialyzed with chelex, before use. To remove
residual cationic metal ions, 1 mM EDTA was added to the high
salt and elution buffers. To determine the dissociation constants
of the metal ions, the initial velocities were repeated using a range
of metal ion concentrations keeping the substrate gluconolactone
concentration at 10 mM. The metal ion dissociation constant
stored at -80 °C prior to purification.
Purification of Human SMP30. All purification proce-
dures were carried out at 4 °C. Frozen cells were thawed and
suspended in a lysis buffer (50 mM Tris-HCl at pH 7.5, 200 mM
NaCl, 5 mM DTT, 1 mM EDTA, 1 mM PMSF, and 10 units/mg
DNase-I) and disrupted by sonication. Detergent extraction was
done immediately in 0.1% (v/v) triton X-100 for 15 min, and the
lysate was centrifuged at 12,000g for 35 min. Supernatant was
incubated with glutathione sepharose 4B (GE Healthcare) beads
for 50 min, flow through was discarded, and the resin was washed
three times with high salt wash buffer (25 mM Tris-HCl at pH
7.5, 1 mM CaCl2, 1 mM DTT, and 170 mM NaCl). The GST-
SMP30 fusion protein was eluted with elution buffer (10 mM
reduced glutathione, 25 mM Tris-HCl at pH 7.5, 1 mM CaCl2,
1
mM DTT, and 170 mM NaCl), concentrated with centrifugal
membranes with a 10 kDa cutoff (Millipore), and was subjected

3
438 Biochemistry, Vol. 49, No. 16, 2010
Chakraborti and Bahnson
(
K ) was quantified by nonlinear fitting of the data to eq 1
RESULTS
We determined the crystal structure of human SMP30 with
d
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2þ
2þ
2
Ca or Zn bound at its active site. The six-bladed β-propeller
ðE þ K
d
þ MÞ - ðE þ K
d
þ MÞ - 4EM
2þ
R ¼ R
f
ð1Þ
fold and active site are shown for the Ca bound form in
2E
2þ
Figure 2. Initially, the crystal structure of Ca -SMP30 was
solved by molecular replacement using a molecular replacement
search model made using a template of a putative lactonase
protein from Agrobaterium tumefaciens, which has 20% protein
using the program GraphPad Prism 3.0, where R and R are the
experimentally determined rate and a floating value of rate
f
(
automatically calculated by the program during curve fitting),
2þ
sequence identity with the SMP30 sequence. The Ca -SMP30
respectively. Since we are fitting the observed enzymatic rates at
various metal concentrations and saturating substrate, this
˚
structure was refined against data to a resolution of 1.4 A, with
final R-factor values of 0.12 and 0.19 for Rwork and Rfree
,
treatment allows us to determine the metal K of the substrate-
d
respectively. This structure enabled a structure solution of the
bound form of the enzyme-metal complex. E and M are the total
enzyme and added metal ion concentrations, respectively, and Kd
is the metal ion dissociation constant.
2þ
Zn -bound form of SMP30 using diffraction data to a resolu-
˚
tion of 1.9 A with final R-factor values of 0.22 and 0.25 for R
work
and Rfree, respectively. For each structure, there are two protein
molecules per asymmetric unit and one metal ion bound to each
protein molecule. The structures were validated by the programs
Procheck and SF check of the CCP4 program suite (23) and the
program WHAT IF (27). A summary of the data collection and
refinement of the crystal structures is presented in Table 1.
Overall Structures of SMP30. The overall structure of
SMP30 contains 24 β-strands forming six β-sheets (Figure 2A,B)
that form a closed circular arrangement around a central solvent
filled tunnel that has one metal binding site. Each β-sheet can be
thought of as a blade of the 6-bladed β-propeller fold. In the
majority of previously solved β-propeller proteins, the active site
is located in this central tunnel. Two regions of higher disorder,
encountered while solving the crystal structures, were assigned to
a flexible one-turn R-helix (residues 268-275) connected with
short loops and a six residue flexible loop (residues 121-127),
which is positioned at the top of the central tunnel presenting a lid
structure to the putative active site. This unique positioning of the
loop may play a role as a gatekeeper to the active site or present
interactions that dictate substrate specificity. The C-terminal tail
of SMP30 is 12 residues long (residues 287-299), and it traverses
across the bottom of the central tunnel. The residues surrounding
the front of the metal binding site (R101, M118, I34, and D104)
form the putative active site of the enzyme (Figure 2D).
Crystallization of Human SMP30. Although a preliminary
crystallization report was reported by others for human
SMP30 (21), our initial crystallization conditions were screened
by commercial crystallization screening kits (crystal screen 1 and 2
and PEG screen, Hampton research). Initial screening hits were
then further refined to optimize the protein crystal quality.
Crystals were obtained at room temperature (25 °C) at protein
concentrations of 3-5 mg/mL using the hanging drop meth-
od (22). Diffraction quality crystals were grown from a crystal-
lization solution containing 0.1 M Tris buffer at pH 7.8, 38%
PEG 6000, 5 mM CaCl , and 5 mM DTT. Aliquots of 2 μL of
2
protein and crystallization solutions were mixed to form each
hanging drop, and crystals formed in 3-7 days. After screening
for cryoprotectant, a suitable stabilizing solution was prepared
containing 0.1 M Tris buffer at pH 7.8, 26% PEG 6000, 30%
PEG 400, 5 mM CaCl , and 5 mM DTT. Crystals were soaked
2
into a stabilizing solution for a few minutes and then flash frozen
in liquid nitrogen and stored in liquid nitrogen until data
collection. Since initial trials to grow crystals with zinc instead
of calcium in the crystallization solution were unsuccessful, we
soaked the calcium-grown crystals in the stabilizing solution
(
without calcium) with a gradual increase in the zinc concentra-
tion up to a final concentration of 1 mM. At ZnCl concentra-
2
tions above 1 mM, the crystals began to crack. One data set was
˚
Single Metal Binding Site. The single metal binding site
observed near the active site is coaxial with the central tunnel of
collected from such a zinc soaked crystal that diffracted to 1.9 A.
X-ray Data Collection and Structure Determination.
Crystals were shipped to the Advanced Photon Source (APS)
of Argonne National Laboratory, Chicago IL through the
express crystallography program. The SGX-CAT beamline was
an automated protein crystal data collection program operated
by SGX Pharmaceuticals. This beamline is presently operated by
Eli Lilly. Data sets were collected at beamline 31-ID-D of the
APS facility. The diffraction data were indexed and processed by
the programs MOSFLM, SCALA, and TRUNCATE of the
CCP4 suite of programs (23).
2þ
the β-propeller. In the Ca -bound crystal structure of SMP30,
2þ
the Ca atom coordinates with residues E18, N154, and D204
2þ
and three water molecules (Figure 2C). The average Ca to
˚
˚
oxygen distances in subunits A and B are 2.13 A for E18, 2.30 A
˚
˚
˚
˚
for N154, 2.18 A for D204, and 2.25 A, 2.28 A, and 2.32 A for the
three water molecules. The residue N103 is also present in close
proximity to this site, though not in direct coordination. In the
2þ
Zn -bound form of SMP30, the residues involved in coordina-
tion with the single Zn atom are identical to those for the Ca
bound form. The average distances in subunits A and B between
2þ
2þ
-
The phases for SMP30 were solved using a molecular replace-
ment search model made using a template (protein data bank
2þ
˚
Zn and each coordinated oxygen atom are as follows: 2.12 A
2
þ
˚ ˚
for E18, 2.27 A for N154, and 2.37 A for D204. The Zn water
(
PDB) code: 2ghs), which shows 20% protein sequence identity
˚
˚
˚
with the SMP30 sequence. Molecular replacement was per-
formed using the program PHASER (24). The top molecular
replacement solution from PHASER was fed into the model-
building program ARP/wARP (23), and it successfully built 551
of the 598 residues in the asymmetric unit. After further cycles of
refinement and loop building by the programs Refmac5 and
COOT (25), a complete model was obtained. Further refinement
was done using the program SHELXL (26) to get the final model
of SMP30.
distances are 2.46 A, 2.57 A, and 2.60 A in chain B, and a single
˚
water was modeled into chain A with a distance of 2.24 A from
2
þ
the Zn atom.
To determine whether the metal ion has a structural or
catalytic role, we did alanine scanning mutagenesis of the metal
residues. The residues E18, N103, N154, and D204 were indivi-
dually mutated to alanine, and the four mutants were expressed
and purified to homogeneity. Relative to the wild type specific
activity, the metal site mutants each displayed a significant loss of

Article
Biochemistry, Vol. 49, No. 16, 2010 3439
2
þ
F
IGURE 2: Crystal structure of human SMP30 with Ca bound. (A) The ribbon structure of SMP30 displays the six-bladed β-propeller fold with
2þ
each blade displayed in a rainbow color. The active site Ca is shown in the middle of the β-propeller as a purple sphere. (B) A 90° x-axis rotation
of the viewin panel A displayingthe loops which project upfrom the β-sheets and definethe surface around the active site. The active site islocated
just above the location of the Ca in this view. (C) The metal binding site of human SMP30 with an electron density map (coefficients 2F
2.0σ) around highlighted residues. The residues E18, D204, and N154 are bound to the Ca . Three water molecules complete the ligation of the
hexa-coordinate Ca . The residues N103 and D104 are near the Ca , but not within bonding distance. (D) The active site residues of SMP30 are
shown. In addition to residues surrounding the Ca (also shown in panel C), three additional residues whose side chains are exposed to the
putative-substrate binding pocket are shown (I34, R101, and M118).
2þ
o
c
- F ,
2þ
2þ
2þ
2þ
2
þ
activity (Figure 3). This assay was performed at a Zn con-
respectively. Although the SMP30 residues E18, N154, and D204
are conserved in all four proteins, there are no additional residues
coordinating with the catalytic metal ion, unlike the structural
homologues PON1, DFPase and XC5397. However, the residue
N103 of SMP30 is present at a position nearly identical to that of
N168 of PON1, and the Asn residue of the other homologues.
The side chain of N103 of SMP30 has a unique conformation
distinctly different from its structural homologues. The chi1 angle
of the side chain is rotated 90° relative to a position that would be
able to coordinate with the metal ion. It might be possible that
this unique positioning of the N103 residue allows a unique
conformation suitable for substrate binding and catalysis. It may
also partly explain how SMP30 binds a variety of divalent metal
ions at the same site, unlike its structural homologues, that
apparently only bind calcium.
centration which was 10-fold greater than the K measured for
the wild type enzyme. The loss of activity seen for these alanine
mutations is interpreted to result from a reduced affinity of the
d
2þ
mutants for the Zn ion, which is essential for catalysis. The
reported values of relative activity are the average of at least two
independent experiments performed in triplicate.
Homology to Other Six-Bladed β-Propellers. A structural
alignment is shown in Supporting Information Figures 1 and 2 of
SMP30 with known six-bladed β-propeller enzymes. SMP30
displayed structural homology with the following proteins:
bacterial drug resistant protein from S. aureus (Drp35), PON1,
DFPase, and a protein from Xanthomonas campestris (XC5397)
(
1
31) with root mean squared deviation (rmsd) values of 2.14, 2.9,
.9, and 1.74 A, respectively. A structural comparison of the
˚
metal binding site of SMP30 with that of Drp35, PON1, DFPase,
and XC5397 reveals similar coordination geometries. The resi-
dues E18, N154, and D204 in SMP30 correspond to E53, N224,
and D269 in PON1; E48, N185, and E236 in Drp35; E21, N175,
and E229 in DFPase; and E48, N191, and D242 in XC5397,
Our preliminary enzyme kinetic studies showed that adding
2þ
2þ
Zn to an assay reaction of the enzyme, purified with Ca
present, showed a marked increase in the reaction rate. This led to
2þ
our pursuit of a crystal structure with Zn bound. We wanted to
2þ
2þ
sort out whether the Zn ion displaced the Ca and bound to

3
440 Biochemistry, Vol. 49, No. 16, 2010
Chakraborti and Bahnson
2þ
the same metal binding site. The structure of the Ca -SMP30
was superposed with the Zn -SMP30 with a rmsd of 0.24 A, and
the metal binding site was nearly identical, as described above
Table 1: Data Collection, Phasing, and Refinement Statistics of SMP30
2þ
˚
2þ
2þ
Zn
Ca
(
Supporting Information Figure 2). To confirm the placement of
Crystal Parameters
Zn at the metal binding site of SMP30, we generated an
anomalous difference Fourier map using anomalous diff-
erence data from the Zn-bound structure of SMP30 collected
at 0.9793 A compared to the Zn-edge of 1.2837 A. The electron
density map was generated using the difference of Bijvoet pairs
for amplitude and phases from the final refined Zn-bound
structure. As shown in Supporting Information Figure 3, a
strong signal is displayed for an anomalous difference Fourier
space group
P21
P21
unit cell dimensions
64.47, 51.00, 86.12
100.11
64.36, 52.01, 85.83
100.11
˚
a, b, c (A), β (deg)
˚
˚
Beamline
SGXCAT 31-ID-D
SGXCAT 31-ID-D
Data Collection
0.9793
˚
wavelength (A)
0.9793
˚
resolution (A)
18.4-1.41
26.0-1.90
(2.02-1.90)
99.0 (94.0)
7.0 (6.3)
a
(
1.49-1.41)
˚
map generated with a 4.0 sigma cutoff. The Ca-edge is at 3.0704 A
and would not display this signal. Our structures unequivocally
completeness (%)
redundancy
I/σI
98.7 (91.9)
7.2 (6.3)
13.4 (3.1)
0.059
2þ
2þ
show that Zn displaces Ca to occupy the same site and does
not occupy a secondary site, confirmed by the lack of a second
peak in the anomalous difference Fourier map within one subunit
of SMP30.
Role of Divalent Cations in Gluconolactonase Activity.
Enzyme kinetic studies of SMP30 with divalent metal ions
showed some interesting results. Although maximum glucono-
10.2 (1.7)
0.083(1.18)
R
merge linear
Refinement Statistics
refinement program
SHELXL
8.0-1.42
0.122/0.191
4703
REFMAC5
26.0-1.92
0.22/0.25
4745
˚
resolution (A)
b
R
work/Rfree
number of atoms
non-hydrogen)
2þ
(
lactonase activity was observed in the presence of Zn , we
observed significant gluconolactonase activity in the presence of
B-factor main chain
B-factor side chain
21.07
29.25
0.01
42.1
45.3
0.03
2.5°
b
2þ
2þ
2þ
Ca , Mg , and Mn as well. The kinetic parameters kcat and
KM of the SMP30 gluconolactonase reaction in the presence of
various metal ions are reported in Table 2. To understand the
physiological role of various divalent cations in the SMP30
reaction, we also calculated the dissociation constants of the
divalent cations toward human SMP30, with respect to the
˚
rmsd bond lengths (A)
c
˚
rmsd bond angles
0.03 A
a
Values in parentheses are for the highest resolution shell.
- I |/Σ(I ), where I is the observed intensity and I is the average
intensity, the sums being taken over all symmetry related reflections.
R-factor = Σ|F - F |/Σ(F ), where F is the observed amplitude and
is the calculated amplitude. Rfree is the equivalent of Rworking, except it is
merge
R =
Σ|I
o
a
a
o
a
o
c
o
o
F
c
2þ
activity of the enzyme. Here, the Mn -bound enzyme showed
maximum affinity (lowest K value) when compared to that of
calculated for a randomly chosen set of reflections that were omitted (5%)
c
from the refinement process (41). The rmsd bond angles are reported as an
d
angle distance using the program SHELXL and as degrees in the program
REFMAC5.
the other cations (Figure 4 and Table 2).
DISCUSSION
In recent years, there has been considerable interest in the
structure and function of SMP30, with several implications of its
antiaging role, predominantly through calcium regulation and
homeostasis. It is also interesting to find out that the SMP30 gene
is highly conserved among eukaryotes (Supporting Information
Figure 4). At the same time, studies have shown that SMP30 is an
enzyme that catalyzes a lactonase reaction on physiologically
occurring substrates such as gulonolactone or synthetic OP com-
pounds such as DFP. It was clear that a crystal structure would aid
in the understanding of the function of this enzyme. On our way
toward solving the first crystal structures of the human SMP30, we
also characterized enzyme kinetics that varies on the basis of which
metal ion is bound at the active site. A comparison of the structural
homologues and the metal dependent studies is discussed further.
A 3D BLAST (28) search with SMP30 identified a bacterial
homologue from Agrobacterium tumefaciens (PDB code: 2ghs)
F
IGURE 3: Relative specific activity of wild type versus E18A,
N103A, N154A, and D204A is shown for the hydrolysis of glucono-
lactone under standard assay conditions described in the Materials
and Methods section with 75 μM Zn
2þ
.
a
Table 2: Kinetic Characterization of SMP30 with Gluconolactone and Its Metal Dependence
a
-1 a
-1 -1 a
b
c
K
M
(mM)
k
cat (s
)
k
cat/K
M
(mM
s
)
K
d
(μM)
cellular concentrations
fM-nM
0.1-10 μM
500 μM
2
þ
þ
þ
Zn
2.7 ( 0.5
0.6 ( 0.1
1.3 ( 0.3
3.7 ( 0.7
341 ( 35
79 ( 8
31 ( 4
48 ( 6
126 ( 27
132 ( 26
24 ( 7
7.0 ( 0.9
0.6 ( 0.1
82 ( 21
2
Mn
2
Mg
2þ
Ca
13 ( 3
566 ( 249
0.1-1 μM (resting)/1000 μM (stress)
a
M
K and kcat values are reported for human SMP30 using the substrate gluconolactone and a saturating concentration of the divalent metal ion listed using
b
d
conditions detailed in Materials and Methods. The reported K values were obtained using 10 mM gluconolactone and varying the metal concentration to
c
obtain velocities. A fit of eq 1 to data shown in Figure 4 yielded the reported values of K
concentration of divalent cations were taken from the literature (34-40).
d
as detailed in Materials and Methods. Estimates of physiological

Article
Biochemistry, Vol. 49, No. 16, 2010 3441
2þ
Ca -dependent lactonase activity. Three of the four conserved
calcium-coordinating residues E48, N191, and D242 of XC5397,
as well as E48, N185, and D236 of Drp35, correspond to E18,
N154, and D204 of SMP30. Although the bacterial enzymes
Drp35 and XC5397 have both been characterized as lactonases,
they have significant differences from SMP30. XC5397 acts as a
dimer and has a total of six calcium ions, two in each subunit and
two in the dimer interface; in contrast, SMP30 has only one metal
per molecule and functions as a monomer. Unlike XC5397,
which can hydrolyze only gluconolactone, SMP30 can hydrolyze
a range of lactones (16). Mouse SMP30 also has been shown to
2þ
display a DFPase activity in the presence of Mg (18). Further-
more, the gluconolactonase activity of SMP30 displayed max-
2þ
imum activity in the presence of Zn , whereas XC5397 required
2þ
Ca for maximum activity (30). Though the hydrolysis of
gluconolactone seems to be an overlapping kinetic function
between XC5397 and SMP30, it is farfetched to correlate the
functions of the bacterial enzyme XC5397 with those of the
mammalian SMP30s. Likewise, a comparison of the active site
residues of the bacterial enzyme Drp35 with SMP30 showed a few
residues that were placed in similar positions and a few others,
which were not. The residues F153, L105, S237, and A90 of
Drp35 correspond well with M118, R101, and G205 of SMP30.
In contrast, the residues N299, F64, D152, and A90 of the Drp35
active site pocket do not have similar residues in SMP30. This
suggests that SMP30 is distinctly different from Drp35 with its
F
IGURE 4: Metal dependent gluconolactonase activity of SMP30 is
2
þ
2þ
2þ
2þ
compared for Zn (9), Mn (2), Mg (0), and Ca (b) bound
enzyme. The gluconolactonase assay was performed with saturating
gluconolactone (10 mM) with enzyme that had been metal depleted,
then incubated with varying concentrations of the indicated divalent
cations. Other details of this comparative assay are described in the
Materials and Methods section.
with the closest matched structure (score of 77 bits). Although the
structure of this protein is available in the PDB, further details are
not yet published. Earlier, we had identified this protein structure
as a homologue, and we used it to construct a search model for
molecular replacement to solve the structure of SMP30. The rmsd
of our SMP30 structure aligned with the structure of this bacterial
2þ
Zn -dependent gluconolactonase activity. Although the cataly-
2þ
tic Ca of Drp35 binds to residues similar to those in SMP30,
2þ
another Ca ion is bound on the surface, which is coordinated to
residues T133, S110, D130, Y135, and G112. There are no
corresponding residues found in SMP30. Although each of the
three bacterial lactonases discussed have overall structures simi-
lar to that of SMP30, they have limited utility to understand the
physiological function of mammalian SMP30.
˚
enzyme was 1.39 A with an alignment length of 280 residues.
However, the sequence identity was only 20% over a stretch of
291 residues. High structural homology together with low
sequence homology has been observed for other members of
the β-propeller fold (29). Although the overall structure of the
bacterial enzyme is very similar to that of SMP30, the absence of
a bound metal in the bacterial enzyme is conspicuous. A close
comparison of the metal binding residues of SMP30 and this
structural homologue reveals that the metal coordinating resi-
dues are conserved. Of the three conserved residues, the positions
of N154 and E18 of SMP30 correspond well with that of N170
and E40 of the bacterial homologue. However, the residue D204
of SMP30 and D221 of the bacterial homologue, though present
in roughly the same positions, have different orientations. The
residue D221 is shifted away from the metal binding center,
thereby making it unsuitable for metal coordination. As both the
SMP30 structure (1.4 A) and the bacterial homologue (1.5 A) are
high-resolution structures, we can infer that a shift of D221 may
explain its inability to bind a metal ion. A comparison of the
putative active site pocket residues of SMP30 with that of the
bacterial homologue showed an overlap of similar residues in the
corresponding positions. N103, R101, C16, and M118 of SMP30
correspond well with N125, R123, L38, and M140 of the bacterial
homologue. Despite the absence of a metal bound to this
bacterial homologue, it is the most structurally similar protein
in the PDB to our reported human SMP30 structure.
Of the eukaryotic β-propellers, the structure and function of
SMP30 most resembles those of PON1 and DFPase. The rmsd’s
˚
of SMP30 overlays with PON1 and DFPase are 2.9 A and
˚
1.87 A, respectively. In addition to the known OP hydrolase
activity of PON1 and DFPase, PON1 has been identified as a
lactonase (19, 32). Although the aromatic lactones that PON1
prefers to hydrolyze (32) are different from gluconolactone and
gulonolactone that are known substrates of SMP30, the compari-
son of the active site residues of PON1 with our SMP30 putative
active-site pocket allows us to predict which residues of SMP30
play a role in determining substrate and metal site specificity.
2þ
˚
˚
The conserved PON1 catalytic Ca -binding residues E53,
N224, and D269 correspond well to E18, N154, and D204 of
SMP30 (Supporting Information Figure 2). However, PON1 has
two other residues (N168 and N270) that coordinate with the
catalytic calcium. The corresponding residues in SMP30 (N103
and G205) are unable to coordinate with the metal ion. Other
critical active site residues of PON1 are H115 and H134, which
2þ
are in close proximity to the catalytic Ca -binding site. From an
alignment, the corresponding residue R101 of SMP30 is placed in
between these two histidines in such a way that its backbone
corresponds to H134, and its side chain nitrogen atoms are very
close to H115.
Other SMP30 homologues include the bacterial enzymes
XC5397, a Xanthomonas campestris protein with an ability to
hydrolyze
D
-glucono-δ-lactone (30), and Drp35, a drug resistance
The HDL-associated enzyme PON1 is different from SMP30
with some distinctive features. PON1 is not a water-soluble
enzyme like SMP30, and it requires detergents or HDL particles
for its function (33). Additionally, there is a second high affinity
protein capable of hydrolyzing lactones (31), from Staphylococ-
cus aureus (Supporting Information Figure 5). The rmsd of
XC5397 and Drp35 with SMP30 are 1.74 A and 2.14 A,
respectively. Interestingly both these bacterial enzymes have
˚
˚
2þ
Ca -binding site deep in the tunnel of PON1, which is referred to

3
442 Biochemistry, Vol. 49, No. 16, 2010
Chakraborti and Bahnson
as the structural calcium. The residues D169, D54, and backbone
carbonyl of I117 of PON1 coordinate with this structural
calcium. However, with the exception of D104, which corre-
sponds well with D169 of PON1, no other corresponding residues
are present in SMP30. We can unequivocally conclude that
SMP30 does not contain this second structural metal site homo-
logous to PON1 and DFPase. The three helices of PON1 give it a
structure distinct from all other homologues. Helix one is the
secreted signal peptide that helps PON1 to bind to HDL, whereas
helices two and three form a canopy to give a somewhat closed
active site. Interestingly, SMP30 has one R-helix (residues
not treated with metal. However, if the metal was catalytic there
would be no difference in the temperature-dependent activity
curve, as we found to be the case, thereby supporting a conclusion
that the metal is catalytic.
Interestingly, all of the reported crystal structures of the
homologues have only calcium bound to the protein and are
each believed to be calcium dependent enzymes. SMP30, how-
2þ
ever, is thought to be a Zn -dependent gluconolactonase, a
2
þ
2þ
Mg -dependent DFPase as well as a Ca -binding protein. We
2þ
have been successful in solving crystal structures bound to Ca
and Zn . This promiscuity toward various divalent cations
becomes more intriguing as the Zn - and Ca -bound structures
have identical metal binding sites and nearly super imposable
structures (Supporting Information Figures 2). We have calcu-
lated the kinetic constants and found that although Zn has the
highest k for the hydrolysis of gluconolactone, Ca , Mn ,
and Mg also catalyze the reaction, albeit at a slower rate
(Table 2). Although the gluconolactone KM for the Ca
dependent activity is a bit higher (3.7 mM) when compared to
2þ
2þ
2þ
2
68-275) located outside its active site and is connected by a
flexible loop. Also there is an additional flexible loop (residue
21-127) that resembles a flap over the tunnel (Figure 2B).
1
2þ
Together, the helix and the loop of SMP30 may be functionally
analogous to the two helices of PON1 that form a canopy outside
its active site.
The squid neuron enzyme DFPase is another member of the
OP hydrolase enzymes with a six-bladed β-propeller architecture.
2þ
2þ
cat
2
þ
2þ
-
2þ
2þ
Although SMP30 and DFPase have a sequence similarity of
˚
the Zn -dependent activity (2.7 mM), the k for the Ca -
cat
-
1
16%, the rmsd of 1.87 A shows these proteins are structurally
dependent activity (48 s ) is only 14% of that found for the
2
þ
-1
quite similar. Notably, it has been reported that SMP30 has a
Zn -bound form (341 s ).
2þ
DFPase activity, in the presence of Mg (17). As SMP30 and
DFPase can both hydrolyze DFP, it seemed instructive to
compare our structure of SMP30 to the active site of DFPase.
To understand the physiological relevance of this metal
dependent activity, we calculated the dissociation constants of
2þ
the metal ions and found that Mn has the highest affinity for
2þ
2þ
The conserved Ca -coordinating residues of DFPase, E21,
D229, and N175, correspond to E18, E204, and N154 of
SMP30 (Supporting Information Figure 2). The fourth cal-
cium-coordinating residue N120 of DFPase corresponds to
N103 of SMP30 and is not coordinated with the metal ion.
One of the active site residues of DFPase, H287, aligns with C16
of SMP30, whereas the other DFPase residue E37 does not have a
related residue in SMP30. It is interesting to note that the R101 of
SMP30 has no corresponding residues in DFPase. Like PON1,
DFPase also has a second structural calcium that is absent in
SMP30. Residues D232, L273, and H274 coordinate with this
calcium, and no corresponding residues were observed in SMP30.
When comparing the overall structure of SMP30 with DFPase,
we see the absence of the prominent helix in the latter. However,
the C-terminal tail of SMP30 and DFPase traverses the tunnel in
a similar fashion on the side opposite the enzymes’ active sites. A
comprehensive mutagenesis and kinetic characterization of
SMP30 is currently underway to elucidate the SMP30 enzyme
mechanism and its relationship to these similar, yet distinct,
eukaryotic enzymes. From the distant sequence homology,
structural homology, and related functions, we have reason to
believe that SMP30, PON1, and DFPase have evolved from a
common protein.
the enzyme. As a result, the concentration of Mn needed to
catalyze the reaction is lower compared to that of the other metal
ions (Figure 4 and Table 2). Notably, when we compare the
reported free concentrations of these divalent cations in the
cell (34-38), with our experimentally determined dissociation
constants of the metal ions, this promiscuity may have significant
functional implications (Table 2). The free cellular concentration
2þ
of Zn is between nanomolar to femtomolar, and its calculated
Kd for gluconolactonase activity is 7 μM. The free cellular
2þ
concentrations of Zn may not be high enough to be physiolo-
2
þ
gically significant. The experimentally determined K of Mn
d
2þ
and Mg are 0.6 μM and 82 μM, respectively, whereas their free
cellular concentrations are roughly 0.1 μM-10 μM and 500 μM,
respectively. Comparing these values to our experimentally
2þ
2þ
determined K values, it is evident that both Mn and Mg
d
could comprehensibly bind to SMP30 and catalyze the glucono-
lactonase reaction in a normal healthy liver or kidney cell.
Apart from the first crystal structures of the human enzyme,
the most exciting and novel aspect of this research is that we have
2þ
established that SMP30 has Ca dependent gluconolactonase
activity. This directly contradicts previous studies that conclude
that SMP30 did not show any enzymatic activity in the presence
-
1
of calcium ions (16, 18). Our measured k value of 48 s
cat
2þ
Human SMP30 contains only one metal bound per protein
subunit as revealed in our crystal structures. This becomes
interesting when compared to the structural homologues de-
scribed previously, which have at least two metal ions per
molecule, one catalytic and the other structural. Our preliminary
site-directed mutagenesis studies on the metal-coordinating re-
sidues demonstrated that the metal ion in SMP30 is catalytic
(Table 2) shows that the Ca -bound enzyme can catalyze the
reaction at a reasonable rate, contrary to previous reports (16).
2þ
Our calculated Kd of Ca
(566 μM) provides a possible
explanation of why the previous studies did not observe any
2þ
activity with calcium ions if conditions with sufficient Ca were
not tried. Alternatively, the previous samples might not have
been metal-free and might have had other high-affinity divalent
cations already bound to the metal binding site. In a separate
(
Figure 3). To further probe the catalytic role of the residues, we
2þ
calculated the temperature dependent activity of the metal-free
enzyme preincubated with or without the metal ions (Supporting
Information Figure 6). If the metal ion was structural, incubation
of the enzyme with high concentrations of calcium, prior to
exposure to higher temperatures, would be expected to retain
wild type activity to a greater extent than the enzyme which was
study, the association constant of Ca has been reported for the
5
-1
rat liver protein to be 4.19 ꢀ 10 M (equivalent to a K of 2.4 ꢀ
d
-
6
10 M) with 6 or 7 calcium binding sites per molecule (13).
However, our crystal structure clearly shows that there is only
one metal binding site in SMP30. The difference between our
calculated K values for Ca (566 ꢀ 10 M) and that of the
2þ
-6
d

Article
Biochemistry, Vol. 49, No. 16, 2010 3443
-
6
earlier study (2.4 ꢀ 10 M) might be because of the different
experimental approaches. While they measured the association
constant by equilibrium dialysis, which does not involve any
manner. This is interesting, as normally the coordination of zinc
is quite distinct from that of calcium in enzymes. It is possible that
the metal coordination is different for the calcium and zinc bound
sites only in the presence of the substrate and thus contributes to
the difference in enzymatic rate. Our studies show for the first
time that the human SMP30 has Ca -dependent enzyme
activity, and differences to previous studies were discussed. We
also compared the physiological concentrations of divalent
substrate, we calculated the K with respect to the enzymatic
activity. We are interested in the role of metal ions with respect to
its gluconolactonase activity and hence developed an assay to
calculate the K in the presence of the substrate, gluconolactone.
Hence, this may explain why our K values are higher than those
d
2þ
d
d
that have been reported previously (13). However, in our case
heterologous expression and purification yielded highly pure
SMP30 protein. The presence in previous studies of trace
quantities of impurities with very high calcium binding affinity
might cause problems when proteins are purified directly from
tissues. An impurity with a high calcium affinity could give
anomalous results.
cations with our calculated metal ion K values and conclude
d
2þ
2þ
that Mn and Mg are the likely functional metal ions for
healthy cells. Overall, our kinetic and structural insights of
human SMP30 allow further elucidation of the physiological
role of this exciting antiaging molecule.
ACKNOWLEDGMENT
To find a possible connection between the gluconolactonase
activity and calcium regulation of SMP30, it is instructive to
Use of the Advanced Photon Source at the Argonne National
Laboratory was supported by the U.S. Department of Energy,
Office of Science, Office of Basic Energy Sciences, under contract
DE-AC02-06CH11357. Use of the SGX Collaborative Access
Team (SGX-CAT) beamline at Sector 31 of the Advanced
Photon Source was provided by SGX Pharmaceuticals, Inc.,
who constructed and operated the facility. We thank Dr. Randy
Read of Cambridge University and Chavas Leo of University of
Manchester for their valuable insights. We also thank Dr. Danny
Ramadan of the Thorpe Lab, for help with the metal binding
analysis.
2þ
compare the experimentally measured K of Ca (566 μM) with
d
that of its free cellular concentrations (0.1-1.0 μM); it is evident
2þ
that Ca is not likely to be the metal of choice for glucono-
lactonase activity in a resting cell. Upon the basis of the numerous
studies on the mouse and rat proteins, one should consider
whether SMP30 functions as an antiaging molecule by maintain-
ing calcium homeostasis. It is well known that the extracellular
free calcium concentration (1-2 mM) is about 10,000-fold higher
than the intracellular concentration (0.05-1.0 μM) as has been
reported extensively in the context of calcium homeostasis and
aging (39, 40). The aging process and reduced cellular function
have been linked to, among other factors, alterations in the
SUPPORTING INFORMATION AVAILABLE
2þ
capacity to maintain a constant low level of Ca . The age related
loss of critical calcium regulatory channels and pumps has been
reviewed extensively (39, 40). It might be possible that a gradual
increase in intracellular calcium with gradual decrease of cal-
cium-pumping functions may alter the calcium concentrations
during aging. If the concentrations of intracellular calcium
Six-bladed β-propeller folds for structural homologues of
human SMP30; superposition of calcium/zinc structures of
SMP30 and with homologues PON1 and DFPase; anomalous
difference Fourier map of Zn-bound SMP30; amino acid se-
quence alignment of eukaryotic versions of SMP30; structure-
based amino acid sequence alignment of human SMP30 struc-
tural homologues prepared with the program STAMP; and
temperature dependent activity curve of human SMP30. This
material is available free of charge via the Internet at http://pubs.
acs.org.
increase above the K value (566 μM), then as per our data,
d
2þ
SMP30 might function as a Ca dependent gluconolactonase
(
Table-2). Also, since all of the age-related studies have been
reportedly done with rodents (which produce vitamin C), a
definitive role of calcium dependence in human SMP30 function
could only be established after further complementary and
supplementary in vivo studies. Regardless, one can conclude that
SMP30 plays an important role in humans due to its high degree
of conservation in the SMP30 protein sequence among higher
eukaryotes (Supporting Information Figure 4).
REFERENCES
1
. Fujita, T., Uchida, K., and Maruyama, N. (1992) Purification of
senescence marker protein-30 (SMP30) and its androgen-independent
decrease with age in the rat liver. Biochim. Biophys. Acta 1116, 122–128.
2. Feng, D., Kondo, Y., Ishigami, A., Kuramoto, M., Machida, T., and
Maruyama, N. (2004) Senescence marker protein-30 as a novel
antiaging molecule. Ann. N. Y. Acad. Sci. 1019, 360–364.
However, the promiscuous nature of metal ions and the high
K values for calcium might actually suggest that SMP30 might
d
3
. Fujita, T., Inoue, H., Kitamura, T., Sato, N., Shimosawa, T., and
Maruyama, N. (1998) Senescence marker protein-30 (SMP30) rescues
cell death by enhancing plasma membrane Ca(2þ)-pumping activity
in Hep G2 cells. Biochem. Biophys. Res. Commun. 250, 374–380.
. Ishigami, A., Fujita, T., Handa, S., Shirasawa, T., Koseki, H.,
Kitamura, T., Enomoto, N., Sato, N., Shimosawa, T., and Maruyama,
N. (2002) Senescence marker protein-30 knockout mouse liver is
highly susceptible to tumor necrosis factor-alpha- and Fas-mediated
apoptosis. Am. J. Pathol. 161, 1273–1281.
function indirectly to regulate calcium levels or have a totally
different function other than calcium regulation and home-
ostasis, especially in the haplorrhini suborder of primates who
are unable to produce their own vitamin C. There might be a yet
unknown alternate physiological substrate that SMP30 will
hydrolyze with a much higher rate in the presence of calcium.
In light of the metal binding studies done previously (13), it will be
4
5
6
. Ishigami, A., Fujita, T., Inoue, H., Handa, S., Kubo, S., Kondo, Y.,
and Maruyama, N. (2005) Senescence marker protein-30 (SMP30)
induces formation of microvilli and bile canaliculi in Hep G2 cells.
Cell Tissue Res. 320, 243–249.
. Ishigami, A., Kondo, Y., Nanba, R., Ohsawa, T., Handa, S., Kubo,
S., Akita, M., and Maruyama, N. (2004) SMP30 deficiency in mice
causes an accumulation of neutral lipids and phospholipids in the liver
and shortens the life span. Biochem. Biophys. Res. Commun. 315, 575–
useful to calculate the calcium K in the absence of substrate to
d
see if the substrate influences this value.
2þ
In conclusion, along with the first high-resolution Ca -bound
2þ
and Zn -bound crystal structures of the human enzyme our
studies show several novel and interesting features of SMP30
never reported before. There is only one metal binding site, which
is quite different from any of its homologues. Also, both zinc and
calcium bind to the same metal-binding site in an identical
580.
7. Maruyama, N., Ishigami, A., Kuramoto, M., Handa, S., Kubo, S.,
Imasawa, T., Seyama, K., Shimosawa, T., and Kasahara, Y. (2004)

3
444 Biochemistry, Vol. 49, No. 16, 2010
Chakraborti and Bahnson
Senescence marker protein-30 knockout mouse as an aging model.
Ann. N. Y. Acad. Sci. 1019, 383–387.
23. CCP4. (1994) The CCP4 Suite: Programs for Protein Crystallogra-
phy, Acta Crystallogr. D Biol. Crystallogr. 50, 760-763.
8
. Son, T. G., Kim, S. J., Kim, K., Kim, M. S., Chung, H. Y., and Lee, J.
(2008) Cytoprotective roles of senescence marker protein 30 against
intracellular calcium elevation and oxidative stress. Arch. Pharmacal
Res. 31, 872–877.
24. Mccoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D.,
Storoni, L. C., and Read, R. J. (2007) Phaser crystallographic soft-
ware. J. Appl. Crystallogr. 40, 658–674.
25. Emsley, P., and Cowtan, K. (2004) Coot: model-building tools for
molecular graphics. Acta Crystallogr., Sect. D 60, 2126–2132.
26. Sheldrick, G. M., and Schneider, T. R. (1997) SHELXL: high-
resolution refinement. Methods Enzymol. 277, 319–343.
9
. Son, T. G., Zou, Y. N., Jung, K. J., Yu, B. P., Ishigami, A.,
Maruyama, N., and Lee, J. (2006) SMP30 deficiency causes increased
oxidative stress in brain. Mech. Ageing Dev. 127, 451–457.
1
1
0. Tuder, R. M. (2006) Aging and cigarette smoke: fueling the fire. Am.
J. Respir. Crit. Care Med. 174, 490–491.
27. Vriend, G. (1990) WHAT IF: A molecular modeling and drug design
program. J. Mol. Graphics 8, 52–56.
28. Altschul, S. F., G, W., Miller, W, Myers, E. W., and Lipman, D. J.
(1990) Basic local alignment search tool. J. Mol. Biol. 215, 403–410.
29. Jawad, Z., and Paoli, M. (2002) Novel sequences propel familiar folds.
Structure 10, 447–454.
30. Chen, C. N., Chin, K. H., Wang, A. H. J., and Chou, S. H. (2008) The
first crystal structure of gluconolactonase important in the glucose
secondary metabolic pathways. J. Mol. Biol. 384, 604–614.
31. Tanaka, Y., Morikawa, K., Ohki, Y., Yao, M., Tsumoto, K.,
Watanabe, N., Ohta, T., and Tanaka, I. (2007) Structural and
mutational analyses of Drp35 from Staphylococcus aureus: a possible
mechanism for its lactonase activity. J. Biol. Chem. 282, 5770–5780.
32. Khersonsky, O., and Tawfik, D. S. (2005) Structure-reactivity studies
of serum paraoxonase PON1 suggest that its native activity is
lactonase. Biochemistry 44, 6371–6382.
1. Yumura, W., Imasawa, T., Suganuma, S., Ishigami, A., Handa, S.,
Kubo, S., Joh, K., and Maruyama, N. (2006) Accelerated tubular cell
senescence in SMP30 knockout mice. Histol. Histopathol. 21, 1151–1156.
2. Yamaguchi, M., Mori, S., and Kato, S. (1988) Calcium-binding
protein regucalcin is an activator of (Ca2þ-Mg2þ)-adenosine tripho-
sphatase in the plasma membranes of rat liver. Chem. Pharm. Bull.
1
(
Tokyo) 36, 3532–3539.
1
1
1
1
3. Yamaguchi, M. (2000) Role of regucalcin in calcium signaling. Life
Sci. 66, 1769–1780.
4. Yamaguchi, M. (2005) Role of regucalcin in maintaining cell home-
ostasis and function (review). Int. J. Mol. Med. 15, 371–389.
5. Linster, C. L., and Van Schaftingen, E. (2007) Vitamin C. Biosynth-
esis, recycling and degradation in mammals. FEBS J. 274, 1–22.
6. Kondo, Y., Inai, Y., Sato, Y., Handa, S., Kubo, S., Shimokado, K.,
Goto, S., Nishikimi, M., Maruyama, N., and Ishigami, A. (2006)
Senescence marker protein 30 functions as gluconolactonase in
L-ascorbic acid biosynthesis, and its knockout mice are prone to
scurvy. Proc. Natl. Acad. Sci. U.S.A. 103, 5723–5728.
33. Josse, D., Ebel, C., Stroebel, D., Fontaine, A., Borges, F., Echalier,
A., Baud, D., Renault, F., Le Maire, M., Chabrieres, E., and Masson,
P. (2002) Oligomeric states of the detergent-solubilized human serum
paraoxonase (PON1). J. Biol. Chem. 277, 33386–33397.
1
7. Billecke, S. S., Primo-Parmo, S. L., Dunlop, C. S., Doorn, J. A.,
La Du, B. N., and Broomfield, C. A. (1999) Characterization of a
soluble mouse liver enzyme capable of hydrolyzing diisopropyl
phosphorofluoridate. Chem.-Biol. Interact. 120, 251–256.
34. Ash, D. E., and Schramm, V. L. (1982) Determination of Free and
Bound Manganese(Ii) in Hepatocytes from Fed and Fasted Rats. J.
Biol. Chem. 257, 9261–9264.
35. Costello, L. C., Guan, Z. X., Kukoyi, B., Feng, P., and Franklin, R. B.
(2004) Terminal oxidation and the effects of zinc in prostate versus
liver mitochondria. Mitochondrion 4, 331–338.
18. Kondo, Y., Ishigami, A., Kubo, S., Handa, S., Gomi, K., Hirokawa,
K., Kajiyama, N., Chiba, T., Shimokado, K., and Maruyama, N.
(
2004) Senescence marker protein-30 is a unique enzyme that hydro-
lyzes diisopropyl phosphorofluoridate in the liver. FEBS Lett. 570,
7–62.
36. Galeotti, T., Palombini, G., and Vanrossum, G. D. V. (1995) Man-
ganese content and high-affinity transport in liver and hepatoma.
Arch. Biochem. Biophys. 322, 453–459.
5
1
2
2
9. Gaidukov, L., and Tawfik, D. S. (2007) The development of human
sera tests for HDL-bound serum PON1 and its lipolactonase activity.
J. Lipid Res. 48, 1637–1646.
0. Scharff, E. I., Koepke, J., Fritzsch, G., Lucke, C., and Ruterjans, H.
(2001) Crystal structure of diisopropylfluorophosphatase from Loligo
vulgaris. Structure 9, 493–502.
1. Warizaya, M., Kinoshita, T., Yamaoka, M., Shibata, T., Saito, N.,
Nakajima, H., and Fujii, T. (2004) Expression, purification, crystal-
lization and preliminary X-ray diffraction studies of human liver
regucalcin. Acta Crystallogr., Sect. D 60, 2019–2021.
37. Sarisa, N. L., Mervaalaa, E., Karppanena, H., Khawaja, J. A., and
Lewenstam, A. (2000) Magnesium An update on physiological,
clinical and analytical aspects. Clin. Chim. Acta 294, 1–26.
38. Williams, R. J. P. (1982) Free manganese(ii) and iron(Ii) cations can
act as intracellular cell controls. FEBS Lett. 140, 3–10.
39. Fujita, T. (2002) Calcium homeostasis and signaling in aging, in
Calcium Homeostasis and Signaling in Aging (Mattson, M. P., Ed.),
pp 13-26, Elsevier, Amsterdam.
40. Yamaguchi, M. (2002) Impact of aging on calcium channels and
pumps, in Calcium Homeostasis and Signaling in Aging (Mattson, M.
P., Ed.), pp 47-65, Elsevier, Amsterdam.
41. Brunger, A. T. (1992) The free R value: a novel statistical quantity for
assessing the accuracy of crystal structures. Nature 355, 472–474.
22. Jancarik, J., and Kim, S. H. (1991) Sparse matrix sampling: a screen-
ing method for crystallization of proteins. J. Appl. Crystallogr. 24,
4
09–411.