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PROTEINS: Structure, Function, and Genetics 24514-515 (1996)
CRYSTALLIZATION ANNOUNCEMENTS
Crystallization and Preliminary X-Ray
Crystallographic Studies of Ketosteroid Isomerase
From Pseudomonas putida Biotype B
Byung-HaOh,' Suhng Wook Kim,' Seong-EonRyu: Sang-So0Kim: Mi-Kyung Yoon:
and Kwan Yong Choi'
'Department of Life Science, Pohang University of Science and Technology, Pohang, Kyungbuk, 790-784,Korea
(S.); 2Protein Engineering Research Group, Korean Research Institute for Bioscience and Biotechnology, KIST,
Daejon, 305-333, Korea (S.); 3R&D Center, Lucky Ltd., Daejon, 305343, Korea (S.)
ABSTRACT
The A5-3-ketosteroidisomerase from Pseudomonas putida biotype B has
been crystallized. The crystals belong to the
space group P2,2,2, with unit cell dimensions of
a = 36.48 A, b = 74.30 A, c =96.02 A, and contain
one homodimer per asymmetric unit. Native diffraction data to 2.19 A resolution have been obtained from one crystal at room temperature indicating that the crystals are quite suitable for
structure determination by multiple isomorphous replacement. o ISXI Wiley-Liss, Inc.
Key words: ketosteroid isomerase, crystallization, protein-steroid interaction
Many isomerases use proton transfer between the
catalytic residues of the enzyme and the substrate in
order to interconvert isomers.1,2 The A5-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of a variety of A5-3-ketosteroids to A4-3-ketosteroids by a stereospecificintramolecular transfer of
the 4P-proton to the 6P p ~ s i t i o n KSI
. ~ from Comamonas testosteroni (renamed from Pseudomonas testosteroni4),which can live on steroids as a sole carbon
source,has been an object of continuous investigation
for many years5p6 as a prototype for understanding
the isomerization mechanism and the protein:steroid
interactions. The enzyme exists as a highly stable
homodimer in ~ o l u t i o nIt. ~is a very efficient enzyme
showing the second-order rate constant KcatlKm to
be 3.0 x 10' (s-'M-') for 5-androstene-3,17-dione,
the most widely employed substrate.8 Site-directed
mutagenesis
have strongly suggested
residue Tyrl4 as the general acid protonating the
3-carbonyl oxygen of the steroid substrates, and residue Asp38 as a general base abstracting the 4pproton of the substrates during the formation of dienolate intermediate. X-ray crystallographic studies
on C. testosteroni KSI started in 1976 with crystals of
the enzyme that had a n unusually large size for one
0 1996 WILEY-LISS, INC.
unit cell axis (a=b = 65.4& c = 504
presenting
technical difficulties in collectinghigh resolution diffraction data. The multiple isomorphous replacement work with this crystal form led t o an initial
interpretation of the electron density map at 6 A
resolution in 1984.14 Subsequently, a 2.5 A resolution native data set was collected using synchrotron
radiation. However, the 6 structure resisted refinement against the 2.5 A resolution data with unacceptably high crystallographic R-factor (personal
communication). Until now, the coordinates of KSI
from C. testosteroni or from any other organism are
not available in the Protein Data Bank. The lack of
accurate high resolution crystal structures of KSI
and KS1:steroid complexes has been an obstacle in
understanding the fine details of the enzyme mechanism, and in obtaining information on the protein:
steroid interactions a t the atomic level.
Recently, the entire gene encoding a homologous
KSI from P . putida biotype B was cloned and overexpressed in E. coZi.15 The polypeptide of the KSI
from P . putida biotype B is six residues longer than
that of C . testosteroni KSI (125 residues). When
amino acid sequences of the two KSI are aligned, 44
amino acids match identically. 15,16 Among those,
the active site residues of the C. testosteroni KSI,
Asp38 and Tyrl4, are conserved as Asp40 and Tyrl6
in P . putida KSI. When the Asp40 and Tyrl6 of the
P. putida KSI were replaced with asparagine and
phenylalanine, respectively, both purified mutant
enzymes exhibited a profound decrease in the catalytic activity and in the K m value as were observed
in the similar mutant enzymes of the C. testosteroni
KSI.17 Thus, the catalytic mechanisms of the two
KSIs appear to be very similar.
a
Received October 26, 1995; accepted October 27, 1995.
Address reprint requests to Byung-Ha Oh, Department of
Life Science, Pohang University of Science and Technology,
Pohang, Kyungbuk, 790-784, Korea (S.)
515
P. PUTIDA KETOSTEROID ISOMERASE
Since we were able to obtain a large amount of
highly purified P. putidu KSI by employing a n affinity column chromatography,15 we initiated crystallization work in a hope to obtain crystals of the
enzyme that are suitable for high resolution structure determination. Initial crystallization conditions were discovered using the screening method of
sparse matrix sampling,” and subsequently the
condition was modified to obtain fewer and larger
crystals in a droplet. Drops of 2 p1 of protein solution
(20 mg/ml, in a buffer solution containing 20 mM
p-mercaptoethanol and 50 mM TrisCl, pH 7.0) were
mixed in equal volume with the precipitant containing 0.1 M sodium acetate, 0.05 M sodium cacodylate
(pH 6.5), and 23% polyethyleneglycol 400. The mixture on a cover slip was then equilibrated against 1
ml of the precipitant in 24-well tissue culture plates
at 22°C. In less than 2 days, many small crystals
appeared in the droplets. It was quite difficult to
suppress too many nucleations, but after many trials it was found that mixing the protein sample and
the precipitant in a 2:5 volume ratio proved most
useful for obtaining crystals of about 0.1 x 0.1 x 0.2
mm in size. Though relatively small, the crystals
diffracted very well. With one such KSI crystal in a
droplet, diffraction data to 2.19 A resolution (94.7%
= 4.6%) were obtained on a Rigaku
complete, R,
R-AXIS IIc imaging plate system (Rigaku, Tokyo,
Japan) operated at 40 kV and 120 mA using CuKa
radiation at room temperature. Using an auto indexing program provided with the Rigaku data reduction software, and examining the diffraction
data set, we found that the crystals belong to the
orthorhombic space group P2,2,2, with unit cell dimensions of a=36.48 A, b=74.30 A, c=96.02 A.
Given the molecular weight of KSI (14,536 Da), we
calculated that the crystal volume per unit molecular weight (V,) is 2.24 A3Da with a solvent content
of 45.1% by volume1s when one unit cell was assumed to contain eight protomers. Thus, the asymmetric unit of the crystals is very likely to contain
one homodimer of KSI.
The favorable characteristics of P. putidu KSI
crystals, as described above, provides a new opportunity to determine the crystal structure of KSI at
high resolution. In addition, P. putidu KSI contains
three cysteine residues (whereas C. testosteroni KSI
contains none) that are useful in obtaining isomorphous crystals derivatized by mercury-based heavy
atom compounds. The structure determination of P.
putidu KSI by the multiple isomorphous replacement method is in progress.
ACKNOWLEDGMENTS
This work was supported by The Basic Science
Research Fund of Pohang University of Science and
Technology, and in part by the Non-Directed Research Fund, Korean Research Foundation, and by
the Research Center for New Bio-Materials in Agriculture.
REFERENCES
1. Fersht, A.R. “Enzyme Structure and Mechanism.” 2nd
edit. New York Freeman, 1984.
2. Walsh, C. “Enzymatic Reaction Mechanisms.” New York:
Freeman, 1984.
3. Batzold, F.H., Benson, A.M., Covey, D.F., Robinson, C.H.,
Talalay, P. The A5-3-ketosteroid isomerase reaction: Catalytic mechanism, specificity, and inhibition. Adv. Enzyme Regul. 14:243-267, 1976.
4. Tamaoka, J., Ha, D.-M., Komagata, K. Reclassification of
Pseudomonus acidouorans den Dooren de Jong 1926 and
Pseudomoms testosteroni Marcus and Talalay 1956 a s Comamonas acidouorans comb. nov. and Comamonns testosteroni comb. nov., with and emended description of the
genus Comamonas. Int. J . Syst. Bacteriol. 3752-59, 1987.
5. Benisek, W.F., Ogez, J.R., Smith, S.B. Recent studies of
steroid isomerase from Pseudomonas testosteroni and
Pseudomonus putida. Ann. N.Y. Acad. Sci. 346:115-130,
1980.
6. Schwab, J.M., Henserson, B.S. Enzyme-catalyzed allylic
rearrangements. Chem. Rev. 90:1203-1245, 1990.
7. Talalay, P., Benson, A.W. A5-3-Ketosteroid isomerase In:
“The Enzyme VI.” 3rd edit. Boyer, P.D. (ed.). New York:
Academic Press, 1972:591-618.
8. Hawkinson, D.C., Eames, T.C.M., Pollack, R.M. Energetics of 3-0x0-A5-ketosteroid isomerase: Source of the catalytic power of the enzyme. Biochemistry 30:10849-10858,
1991.
9. Xue, L., Talalay, P., Mildvan, A.S. Studies of the mechanism of the A5-3-ketosteroid isomerase reaction by substrate, solvent, and combined kinetic deuterium isotope
effects on wild-type and mutant enzymes. Biochemistry
29:7491-7500, 1990.
10. Kuliopulos, A,, Mullen, G.P., Xue, L., Mildvan, A.S. Stereochemistry of the concerted enolization catalyzed by A53-ketosteroid isomerase. Biochemistry 30:3169-3178,
1991.
11. Xue, L., Kuliopulos, A,, Mildvan, A S , Talalay, P. Catalytic mechanism of a n active-site mutant (D38N) of A5-3ketosteroid isomerase. Direct spectroscopic evidence for dienol intermediates. Biochemistry 30:4991-4997, 1991.
12. Xue, L., Talalay, P., Mildvan, A.S. Studies of the catalytic
mechanism of an active-site mutant (Y14F) of A5-3-ketosteroid isomerase by kinetic deuterium isotope effects. Biochemistry 30:10858-10865, 1991.
13. Westbrook, E.M. Characterization of a hexagonal crystal
form of a n enzvme of steroid metabolism., A5-3-ketosteroid
isomerase: A new method of crystal density measurement.
J . Mol. Biol. 103:659-664, 1976.
14. Westbrook, E.M., Piro, O.E., Sipler, P.B. The 6-A crystal
structure of A5-3-ketosteroid isomerase. J . Biol. Chem.
259:9096-9103, 1984.
15. Kim, S.W., Kim. C.Y., Benisek, W.F., Choi, K.Y. Cloning,
nucleotide sequence, and overexpression of the gene coding
for A5-3-ketosteroid isomerase from Pseudomonas putida.
J. Bacteriol. 21:6672-6676, 1994.
16. Linden, K.G., Benisek, W.F. The amino acid sequence of a
A5-3-ketosteroid isomerase from Pseudomonus putida biotype B. J. Biol. Chem. 261:6454-6460, 1986.
17. Kim, S.W., Choi, K.Y. Identification of active site residues
by site-directed mutagenesis of A5-3-ketosteroid isomerase
from Pseudomonas putida biotype B. J . Bacteriol. 177:
2602-2605, 1995.
18. Jancarik, Y., Kim, S.-H. Sparse matrix sampling: A
screening method for crystallization of proteins. J . Applied
Cryst. 24:409-411, 1991.
19. Matthews, B. Solvent content of protein crystals. J. Mol.
Biol. 33:491-497, 1968.
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