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PROTEINS Structure, Function, and Genetics 24525-527 (1996)
Crystallization and Preliminary X-Ray Diffraction
Study of 1,3&Trihydroxynaphthalene Reductase
From Magnaporthe grisea
Arnold Andersson,' Doug Jordan? Gunter Schneider? Barbara Valent? and n v a Lindqvist3
'Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala Biomedical Center,
S-75124 Uppsala, Sweden; 'Dupont Agricultural Products, Stine-Haskell Research Center, Newark, Delaware
19714, USA; 3Division of Structural Biology, Department of Medical Biochemistry and Biophysics, Karolinska
Institute, S-171 77 Stockholm, Sweden; and 4Central Research and Development, Dupont Experimental Station,
Wilmington, Delaware 19880-0402, USA
reductase was crystallized in the presence of
NADPH and the inhibitor tricyclazole. The
crystals are trigonal, space group P3,21 or its
enantiomorph P3,21. Two crystal forms with
slightly different cell dimensions were obtained. Form A has unit cell dimensions a = b =
142.6 hi, c = 70.1 hi and form B cell dimensions a
= b = 142.6 A, c = 72.9 A. The diffraction pattern of the latter crystal form extends to 2.5 A
resolution. o 1996 Wiley-Liss, Inc.
Key words: naphtol reductase, melanin synthesis, rice blast disease, fungicide, rational drug design, crystallography
Melanin formation by the fungal plant pathogen
Magnaporthe grisea is fundamental to the development of rice blast disease.'s2 The pathogen penetrates the outer plant leaf surface by using a specialized fungal cell called an appressorium. A layer
of melanin is sandwiched between the appressorial
cell wall and plasma membrane; this construction
enables the appressoria to build up hydrostatic pressure. In the later stages of the infection a penetration peg is formed from the appressoria and driven
into the leaf via the high pressure, which gives the
pathogen access to host epidermal cells.
Some M. grisea mutants with deficient melanin
biosynthesis are not pathogenic, since they lack certain key enzymes. Non-pathogenicity is due to a
defective appressoria, which is caused by lack of
melanin, resulting in an inability to build up hydrostatic pressure. Enzymes in the melanin pathway
are thus prime targets for fungicide control. We
have therefore initiated crystallographic studies of
enzymes from this pathway. The crystal structure
of one of these enzymes, scytalone dehydratase in
complex with an inhibitor, has been determined to
2.9 h r e s o l ~ t i o nIn
. ~ this communication we report
on the crystallization of another enzyme from this
pathway, 1,3&trihydroxynaphthalene reductase
THNR from M. grisea has a molecular weight of
120 kDa and consists of four subunits, each of which
contains 282 amino acids? The enzyme converts
1,3,8 trihydroxynaphthalene to vermelone in the
biosynthesis of melanin. The overall reaction is
highly reversible, and the kinetic mechanism is
thought to be ordered with pyridine nucleotide binding to the enzyme before the napthol substrate.
Enzyme Source
THNR was purified from E. coli cells that had
been transformed with plasmids containing the
cDNA coding for THNR under the transcriptional
control of an inducible T7 promoter. The gene for
THNR was isolated from a cosmid library of M.
grisea by complementation of the defective THNR in
a Buf mutant, CP723, and the cDNA corresponding
to the THNR gene was cloned by reverse transcription and polymerase chain reaction (PCR) methodology. Further details on cloning and expression will
be reported elsewhere.
All protein purification steps were at 4°C. Thawed
E. coli cells were diluted 1:4 (w/v) into 25 mM
HEPES-NaOH, pH 7.5 (Buffer H), and 10 Fg/mL
leupeptin. Cells were broken by two passes at 18,000
psi through a microfluidizer (model 11OY; Microfluidics Corp.). Cellular debris was removed by centrifugation for 15 min at 23,OOOg. The supernatant
was brought to 25% saturation with respect to
(NH,),SO, by dropwise addition of a saturated solution. Following a 15 min centrifugation at 40,OOOg
the supernatant was applied to a phenyl-Sepharose
CL-4B (Pharmacia) column equilibrated with Buffer
H and 25% saturated (NH,),SO,. The column was
Received October 7, 1995; accepted October 13, 1995.
Address reprint requests to Ylva Lindqvist, Division of
Structural Biology, Department of Medical Biochemistry
and Biophysics, Karolinska Institute, S-171 77 Stockholm,
subsequently washed with 1 bed volume of equilibration buffer followed by a 5 bed-volume linear
gradient ranging from equilibration buffer to equilibration buffer lacking (NH,),SO,. Fractions with
enzymatic activity were pooled and precipitated by
the addition of (NH,),SO, to give 70% saturation.
Protein was solubilized in a minimum volume of
Buffer H and loaded onto a Toyopearl HW-55s
(Tosohaas) column that was equilibrated and developed and Buffer H. Fractions with enzymatic activity were pooled and loaded onto a Q-Sepharose Fast
Flow (Pharmacia) column equilibrated in Buffer H.
The column was washed with 1bed volume of Buffer
H followed by a 7 bed-volume linear gradient increasing from 0 to 100 mM NaCl in Buffer H. Active
fractions were pooled and loaded onto a column of
2'5' ADP Sepharose 4B (Pharmacia) equilibrated in
Buffer H containing 50 mM NaC1. The column was
washed with a bed volume of equilibration buffer,
and enzyme was eluted by washing the column with
Buffer H containing 1 M NaC1. THNR fractions
were concentrated and diafiltered into Buffer H by
using a pressure cell and a PMlO membrane (Amicon). THNR was homogeneous on sodium dodecyl
sulfate polyacrylamide gel electrophoresis and isoelectric focusing gels. Electrospray mass spectroscopy gave a mass of 29,982 versus a calculated mass
of 29,984 Da. Typically, 0.3 g of homogeneous THNR
was obtained from 70 g (wet weight) of cells.
Crystallization experiments were performed in
the presence and absence of a tenfold excess of the
coenzyme NADPH and the inhibitor tricyclazole (5methyl-l,2,4-triazolo[3,4-~]benzothiazole)
using the
hanging drop technique, by mixing equal volumes (5
~ 1of) protein solution (20 mg/ml in 5 mM HEPES, 5
mM NaC1, pH 7.5) and well solution. Initial screening for crystallization conditions was performed by a
simplified version5 of incomplete factorial experiments6 Initially, 50 different conditions for crystallization were tested at 4°C and 20°C. Optimal crystallization conditions were obtained by subsequent
fine grid screening for those conditions that initially
gave crystals.
X-Ray Crystallographic Studies
The three-dimensional data sets were collected
with a Raxis I1 imaging plate mounted on a Rigaku
rotating anode operating a t 50 kV and 180 mA.
Each frame was recorded as a 1.2" oscillation image
using a 0.3 mm collimator. Data processing was carried out with Denzo7 and CCP4.8 Space group determination was done using the autoindexing option in
Denzo in combination with the analysis of simulated
precession photographs using the program PATTERN (G. Lu, unpublished information).
Two different trigonal crystal forms, grown in the
presence of NADPH and the inhibitor tricyclazole,
were obtained. Both forms grew under the same conditions, 18% polyethylene glycol 6000 at pH 6.6.
These crystals belonged to the space group P3,21 or
its enantiomorph P3,21 but showed slight differences in cell dimensions. One crystal form had cell
dimensions a = b = 142.7 A, c = 70.1 A; these
crystals can grow to a size of up to 1.2 x 1.2 x 0.4
mm. The packing densities were 3.4, 2.3, and 1.7
b31Da for two, three, or four subunits in the asymmetric unit, respectively.' A data set from this crystal form was collected to 3.5 A (kerge
6.4%, 70.9%
The second crystal form had cell dimensions a = b
= 142.6 A, c = 72.9 A, resulting in similar packing
densities. These crystals grew to a size of 0.8 X 0.8
x 0.4 mm, and their diffraction pattern extended to
2.5 b resolution using a conventional X-ray source.
These crystals are stable in the X-ray beam for up to
3 days, and a data set to 3.0 b resolution (kerge
7.6%, 93.4% complete) could be collected from one
The native Patterson function, using data from
the second crystal form in the resolution interval
5-45 A, showed no significant peaks. Self-rotation
function calculations were carried out using the program AMORE at different resolution shells and integration radii." One significant peak (38% of origin) with Eulerian angles ci = go", p = 124.8", y =
90" was found, indicating a non-crystallographic
twofold rotation axis perpendicular to the crystallographic c-axis. In conclusion, crystals of THNR are
suitable for a crystallographic structure determination, and the search for heavy atom derivatives is in
This work was supported by the Magnus Bergvalls Foundation.
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2. Howard, R.J., Ferrari, M.A. Role of melanin in appressorium function. Exp. Mycol. 13:403-418, 1989.
3. Lundqvist, T.,Rice, J., Hodge, N.C., Basarab, G., Pierce,
J., Lindqvist, Y. Crystal structure of scytalone dehydratase-a disease determinant of the rice pathogen, Mugnaporthe griseu. Structure 2:937-944, 1994.
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ceedings of the CCP4 Study Weekend.” Daresbury Laboratory, Warrington, UK, pp. 56-62, 1993.
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