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PROTEINS: Structure, Function, and Genetics 25510-513 (1996)
Crystallization of the Purine Salvage Enzyme Adenine
Phosphoribosyltransferase
Cynthia L. Phillips, Buddy Ullman, and Richard G. Brennan
Department of Biochemistry and Molecular Biolog.y, L-224, Oregon Health Sciences University, Portland, Oregon
97201 -3098
ABSTRACT
Adenine
phosphoribosyltransferase from the protozoan parasite Leishmania domvani has been crystallized in the
presence of the substrate Mg2+-a-~-5-phosphoribosyl-1-pyrophosphate(PRPP)or the product
adenosine-5-monophosphate,as well as in the
absence of ligand. These crystals belong to the
space group P6,22 or its enantiomorph P6,22,
with unit cell dimensions of a = b = 64.0 A, c =
240.5 A, (Y = p = go",and y = 120".The crystals
diffract to 1.9 A. o 1996 Wiley-Liss, Inc.
A high-resolution crystal structure of the L. donovani APRT would be an important first step in the
design of new reagents, providing an atomic framework for structure-based inhibitor or subversive
substrate design?-''
In addition to drug design, the structure of the L.
donovani APRT will permit structural comparison
with the recently solved crystal structures of the hypoxanthine/guanine PRT (HGPRT) from humans,
the orotate PRT (OPRT) from Salmonella typhimurium, and the glutamine PRPP amidotransferase
from Bacillus subtil~s."-'~ Despite their divergent
primary sequences, the tertiary structures of these
Key words: Leishmania, parasite metabolism,
PRTs show a highly conserved core structure that
selenomethionine, X-ray diffracextends to their nearby nucleotide binding f01ds.l~
tion
However, their structural similarities fade toward
the solvent-accessible surfaces, at the oligomerizaINTRODUCTION
tion interfaces, and at the interacting N- and C-terAdenine phosphoribosyltransferase (APRT), a
minal regions and loops, which include their active
member of the phosphoribosyltransferase (PRT)
sites. The structure of L. donovani APRT, which
family, is responsible for recycling the purine base
shares
only 9% sequence identity with human
adenine by catalyzing the Mg2+-dependent converHGPRT,
for example, will define more clearly the
sion of adenine and a-~-5-phosphoribosy1-l-pyrostructural
requirements for catalysis of members of
phosphate (PRPP) to adenosine-5-monophosphate
the
PRT
family
and delineate which regions of the
and pyrophosphate.' In mammals and other organunrelated
primary
sequence comprise these funcisms that can synthesize purines de novo, recycling
tionally
related
structures.
Moreover, a structure of
of purines is primarily important during periods of
an
APRT
will
likely
lend
insight
into the structural
rapid growth, such as for rapidly dividing cancer
basis
for
the
sequence
divergence
found between the
cell^.^,^ Although APRT is not essential for human
human
and
L.
donovani
APRTs,
which
are only 27%
cells, a genetic defect in APRT activity is not withidentical,
yet
catalyze
identical
reactions.
out consequence, causing a 2,8-dihydroxyadenine
Finally, the structure of APRT should contribute
urolithiasis (kidney stone^).^ In contrast, protozoan
to
an understanding of the function of the varied
parasites lack a de novo biosynthetic pathway for
oligomeric
states in which PRTs exist and the role of
purines and must scavenge purines from their host.5
oligomerization
in catalysis. Studies on S. typhimuThese differences in purine metabolism of protozoan
rium OPRT show that this enzyme functions as a
parasites, as well as tumor cells, relative to their
dimer in which Lysine 103 of one monomer is rehuman hosts suggest that enzymes of the purine salquired
for catalytic activity of the other monovage pathway, such as APRT, are judicious targets
mer.15,16
Human HGPRT is also a dimer, but the
for chemotherapeutic intervention.
structural relationship between the oligomeric state
The protozoan parasite Leishmania donovani
and catalysis is unknown. The glutamine PRPP
causes visceral leishmaniasis, a fatal disease if unamidotransferase is a cooperative tetramer. Gel filtreated. Unfortunately, therapies for leishmaniasis
tration
experiments on L. donovani APRT show that
are far from ideal and employ highly toxic and nonselective drugs. APRT appears to be important, if
not indispensible, for purine salvage by the infective
form of the parasite.6 The L. donovani aprt gene has
Received January 19, 1996; accepted February 2, 1996.
Address reprint requests to Richard G. Brennan, Dept. of
been cloned, sequenced, and overexpressed in EsBiochemistry and Molecular Biology, L-224, Oregon Health
cherichia coli, and the recombinant APRT protein
Sciences University, 3181 SW Sam Jackson Park Road, Porthas been purified in large amounts to h~mogeneity.~ land, OR 97201-3098.
0 1996 WILEY-LISS,INC.
511
CRYSTALS OF ADENINE PHOSPHORIBOSYLTRANSFERASE
it behaves as a monomer in the presence of either
Mg2+ -PRPP or AMP.7 Whether there is an internal
catalytic lysine in APRTs that functions analogously to the lysine of S. typhimurium OPRT is not
known. High-resolution structures will be valuable
for a more complete understanding of the general
structure of PRTs both before and after catalysis.
MATERIALS AND METHODS
AND RESULTS
Expression and Purification of
L. donovani APRT
The L. donouani APRT gene encodes a 238 amino
acid protein and has been subcloned into the pBAce
expression vector and overexpressed in the E . coli
strain ,54446, which lacks the bacterial APRT.7 Minor modifications were made to the single-step chromatography purification from Leishmania cell culture described by Allen et al.17 The E . coli cells were
inoculated from a frozen glycerol stock and harvested after 15 hours of growth at 37°C in low phosphate media. The cells were lysed and sonicated in
TMD2O buffer (20 mM Tris, pH 7.4, 5 mM MgCl,, 1
mM dithiothreitol), with 1 mM phenylmethylsulfonyl fluoride and 1pg/ml DNase. The lysate was centrifuged at 20,OOOg for 20 minutes and the supernatant loaded onto an AMP-agarose affinity column
(Sigma, catalog #A-3019) and eluted with 1 mM
PRPP or 2 mM AMP in TMD2O buffer. A typical
yield was 5 mg of purified protein per liter of bacterial cells, and the purity was > 95% as judged by
sodium dodecyl sulfate polyacrylamide gel electrophoresis.
The selenomethionine-incorporated APRT was
overexpressed by transforming the pBAce expression vector directly into the E . coli strain DL41, an
auxotroph for methionine (Yale Genetic Stock Center, strain 7177;h-, metA28). There are six methionine residues in L. donouani APRT, including the
amino-terminal methionine. Growth in modified
"Hail Mary" media (defined media with each amino
acid added separately") was exceedingly slow, with
a doubling time of more than 2 hours. The media
included 0.1 mM K2HP04as the sole source of phosphate, and D/L-selenomethionine at 75 mg/L was
substituted for methionine. A 5 ml culture containing 100 pg/ml methionine was used to inoculate
each liter of selenomethionine-containing media, resulting in a less than 1%dilution of the selenomethionine by methionine. The purification was otherwise identical to that for unlabeled APRT, with a
final yield of 1 mg of APRT per liter of bacterial
culture.
Crystallization and Preliminary X-ray
Characterization of L. donovani APRT
Purified APRT was concentrated to 11 mg/ml in
TMD2O buffer containing either 1 mM PRPP or 2
mM AMP. Protein concentration was determined by
ultra-violet absorbance using a calculated extinction coefficient of
= 16,100 cm-' M-l. Crystals
of native APRT were obtained by vapor diffusion
using both hanging and sitting drops. Initial crystallization screens using Hampton Research Crystal
Screens yielded crystals in 30% of the conditions in
Crystal Screen I and 14% of the conditions in Crystal Screen 11. All but two of these crystal forms appeared to be hexagonal bipyramids. To produce the
highest quality diffraction crystals, the APRT was
mixed 1:l (v:v) with the crystallization reagent [1.2
to 1.6 M (NH,),SO, in 0.1 M Na-Acetate, pH 4.901
and equilibrated in 12 pl drops over 1ml reservoirs
a t 4°C for 2 weeks t o 2 months. Crystals of APRT
were enzymatically active by radioactive assay."
The crystals of selenomethionine-incorporated
APRT grew similarly.
The L. donouani APRT crystals were grown reproducibly to dimensions of 0.5 mm x 0.5 mm x 0.8
mm in the presence of M 8 -PRPP or AMP or in the
absence of ligand. Although the protein had to be
used for crystallization within 1 week of purification, once grown, the crystals were stable for months
and diffract beyond 1.9 A. Precession photographs of
the hkO and Okl planes revealed hexagonal symmetry and a 6, or 6, screw axis. Both substrate and
product crystal forms have unit cell dimensions of
a = b = 64.0A,c = 240.5A,cw = p = 90",andy =
120" and assume space group P6,22 or its enantiomorph P6,22. One monomer per asymmetric unit
yields a VM of 2.73 A3fDa and a solvent content of
+
55%.,O
Data Collection
Initial intensity data of the native apo-APRT crystals or crystals grown in the presence of either
Mg2+-PRPPor AMP were collected a t room temperature using an Area Detector Systems Corporation
(ADSC) multiwire area detector and processed with
software provided by ADSC. The rotating anode
X-ray source (Rigaku RU2OOH) operated a t 40 kV
and 150 mA. Data for APRT crystals grown in the
presence of M8+-PRPP had an R,
of 3.89% at
3.6-21.5
resolution (3,775 unique reflections,
99.0% complete), an R,, of 5.25% at 2.6-5.0 A resolution (7,475 unique reflections, 91.0% complete),
and an Ray, of 7.66% a t 1.95-3.15 A (7,297 unique
reflections, 43.7% complete). Data on APRT crystals
grown in the presence of AMP had an R,
of 4.66%
a t 3.6-21.5 A resolution (3,612 unique reflections,
94.7% complete) and an Ray, of 6.57% a t 2.6-5.0 A
resolution (7,959 unique reflections, 96.8% complete). Data for the apo-APRT crystals had an Rsym
of 3.71% a t 2.3-4.9 A resolution (9,816 unique reflections, 80.2% complete) and an R,, of 4.09% at
3.0-20.0 A resolution (2,681 unique reflections,
95.5% complete). Merged intensity data for the
APRT crystals grown in the presence of Mg2+-PRPP
had an Rsymof 7.8% (21.5-2.6 A); for crystals grown
5 12
C.L. PHILLIPS ET AL.
TABLE I. Derivative Statistics
HgC1,
Resolution (A)
Unique reflections
Completeness (%)
R (%)*
PCMBS
14-3.6
3,648
96.9
3.53
0.206
14-3.6
3,674
97.6
3.39
“7
0.236
RIB0
:R,.
on intensities = Z,, Zi(Ii(hk 1) - I(h k 111 / Z, Zi I,(h k 1).
4, = L l IFderi.Ahk 1)l - IF&i”Jh k 1)l I 1
Thimerosal
CH3HgC1
Selenomethionine
14-3.6
3,679
97.7
3.72
0.232
14-2.6
3,110
82.6
5.03
0.215
14-3.6
3,729
99.0
2.03
0.097
mdivel.
in the presence of AMP, an Rsymof 7.8% (21.5-2.6
A); and apo-APRT had an Rsymof 7.0% (20.0-2.3 A).
It is interesting that, as observed for 0PRT,l5 substrate or product binding does not alter noticeably
the unit cell dimensions.
Heavy Atom Derivatives
Four mercurials, HgCl,, thimerosal, CH,HgCl,
and p-chloromercuriphenylsulfonic acid (PCMBS),
have shown promise as derivatives. Precession photographs of each of these potential derivatives
showed intensity differences from native APRT.
Similar intensity changes for the HgCl, and the
thimerosal-soaked crystals suggested derivatization
a t the same site($. Three-dimensional intensity
data from 15.0-3.6 A have been collected for each of
the mercurial soaked crystals, and all of these appeared to be isomorphous with the native crystals.
The selenomethionine-labeled APRT crystals also
appeared to be isomorphous and were as stable in
the X-ray beam as native APRT. Some statistics relevant to these derivatives are given in Table I. The
mercurials and selenomethionine derivatives are
good candidates for the solution of the APRT structure by standard multiple isomorphous replacement
methods.
SUMMARY
The excellent diffraction quality of the L. donouani APRT crystals suggests that the ensuing structure will provide a high-resolution framework for
structure-based inhibitor and design. Furthermore,
the structural determination of the L. donouani
APRT will contribute to our understanding of its
catalytic mechanism and more generally to PRT
structure both before and after catalysis. Finally,
the L. donouani APRT structure may reveal differences between PRTs from organisms capable of de
novo purine synthesis anti those that rely upon purine salvage.
ACKNOWLEDGMENTS
We thank Darrick Carter for determining the catalytic activity of crystallized APRT and Patrick
Jimenez for initial crysta.llizations. This work was
supported by grants from the Oregon Community
Foundation to R.G.B. and the Public Health Ser-
vices (A1236821 to B.U. C.L.P. is a Burroughs Wellcome Life Sciences Research Fellow. B.U. is a Burroughs Wellcome Fund Scholar in Molecular
Parasitology, and this work was supported in part
by a grant from the Burroughs Wellcome Fund.
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