676
Alterio et al.
tration of 1 mM were carried out at 208C in 20 mM sodium phos-
phate buffer pH 7.4. The hydrolysis of S-derivatives of glutathione
was determined by monitoring the respective decrease in absorb-
ance at 240 nm.
3. Lee, E. Y.; Lee, W. H. Proc Natl Acad Sci USA 1986, 83, 6337–
6341.
4. Degrassi, G.; Uotila, L.; Klima, R.; Venturi, V. Appl Environ
Microbiol 1999, 65, 3470–3472.
5. Yurimoto, H.; Lee, B.; Yano, T.; Sakai, Y.; Kato, N. Microbiology
2003, 149, 1971–1979.
Crystallization and X-Ray Data Collection
6. Herring, C. D.; Blattner, F. R. J Bacteriol 2004, 186, 6714–6720.
7. Gonzales, C. F.; Proudfoot, M.; Brown, G.; Korniyenko, Y.;
Mori, H.; Savchenko, A. V.; Yakunin, A. F. J Biol Chem 2006,
281, 14514–14522.
PhEst was crystallized at 293 K using the hanging drop vapor diffu-
sion technique. Drops were prepared by mixing 1 ll of enzyme solu-
tion (4 mg ml21 in 25 mM TRIS-HCl, pH 7.3) with 1 ll of precipi-
tant solution (20% (w/v) PEG MME 5000, 0.2M sodium acetate,
0.1M TRIS-HCl, pH 7.0), and equilibrated over a well containing 1
ml of precipitant solution. Crystals appeared after 3 days and grew
in about one week to maximum dimensions of 0.3 3 0.2 3 0.2
8. Kordic, S.; Cummins, I.; Edwards, R. Arch Biochem Biophys
2002, 399, 232–238.
9. Haslam, R.; Rust, S.; Pallett, K.; Cole, D.; Coleman, J. Plant
Physiol Biochem 2002, 40, 281–288.
3
˚
mm . A complete dataset was collected at 2.20-A resolution from a
10. Cummins, I.; McAuley, K. M.; Fordham-Skelton, A.; Schwoerer,
R.; Steel, P. G.; Davis, B. G.; Edwards, R. J Mol Biol 2006, 359,
422–432.
single crystal at the temperature of 100 K, with a copper rotating an-
ode generator developed by Rigaku and equipped with Rigaku Sat-
urn CCD detector. Prior to cryogenic freezing, crystals were trans-
ferred to the precipitant solution with the addition of 15% (w/v)
glycerol. Data were processed using the HKL2000 crystallographic
data resolution package (Denzo/Scalepack).47 The crystals belonged
to the space group P212121 with unit cell dimensions of a 5 49.49
11. Legler, P. M.; Kumaran, D.; Swaminathan, S.; Studier, F. W.;
Millard, C. B. Biochemistry 2008, 47, 9592–9601.
12. Wu, D.; Li, Y.; Song, G.; Zhang, D.; Shaw, N.; Liu, Z. J. FASEB J
2009, 23, 1441–1446.
13. van Straaten, K. E.; Gonzalez, C. F.; Valladares, R. B.; Xu, X.; Sav-
chenko, A. V.; Sanders, D. A. Protein Sci 2009, 18, 2196–2202.
14. Aurilia, V.; Parracino, A.; Saviano, M.; Rossi, M.; D’Auria, S.
Gene 2007, 397, 51–57.
˚
˚
˚
A, b 5 129.75 A, c 5 152.67 A. The Matthews coefficient (VM
5
1.99 A Da21) indicated that the crystallographic asymmetric unit
contained four molecules according to a solvent content of 38%.
Data collection statistics are reported in Table I.
3
˚
15. Laskowsky, R. A.; MacArthur, M. W.; Moss, D. S.; Thornton, J.
M. J Appl Cryst 1993, 26, 283–291.
16. Hutchinson, E. G.; Thornton, J. M. Protein Sci 1996, 5, 212–
220.
Structure Determination and Refinement
The structure of PhEst was solved by molecular replacement tech-
nique using the program AMoRe48 and the crystallographic struc-
ture of the ScFGH (PDB code 1PV1)11 as model template. The rota-
tion and translation functions were calculated using data between
17. De Simone, G.; Galdiero, S.; Manco, G.; Lang, D.; Rossi, M.;
Pedone, C. J Mol Biol 2000, 303, 761–771.
18. De Simone, G.; Mandrich, L.; Menchise, V.; Giordano, V.; Feb-
braio, F.; Rossi, M.; Pedone, C.; Manco, G. J Biol Chem 2004,
279, 6815–6823.
˚
15.0 and 3.5 A resolution. The one-body translation search, using
the centred-overlap function (c-o), on the first 50 rotation solutions
led to a single solution with a correlation coefficient of 25.4% and
an R-factor of 52.1%. The n-body translation search carried out
with the phased-translation function (p-t), by including a PC
refinement before each n-body translation search led to finding the
remaining three molecules contained into the asymmetric unit. This
improved the correlation coefficient and the R-factor to 73.8 and
32.7%, respectively. Refinement of the structure was carried out
using CNS23 and model building was performed with O.49 The first
19. Bentahir, M.; Feller, G.; Aittaleb, M.; Lamotte-Brasseur, J.;
Himri, T.; Chessa, J. P.; Gerday, C. J Biol Chem 2000, 275,
11147–11153.
20. Hoyoux, A.; Jennes, I.; Dubois, P.; Genicot, S.; Dubail, F.; Fran-
c¸ois, J. M.; Baise, E.; Feller, G.; Gerday, C. Appl Environ Micro-
biol 2001, 67, 1529–1535.
21. Lonhienne, T.; Zoidakis, J.; Vorgias, C. E.; Feller, G.; Gerday, C.;
Bouriotis, V. J Mol Biol 2001, 310, 291–297.
22. Tina, K. G.; Bhadra, R.; Srinivasan, N. Nucleic Acids Res 2007,
35, W473–W476.
cycles of the refinement were carried out with four-fold NCS-
restraints with an energy barrier of 300 kcal mol21 A . After R-fac-
2
˚
¨
23. Brunger, A. T.; Adams, P. D.; Clore, G. M.; De Lano, W. L.;
tor and R-free reached 21.7 and 24.1%, respectively, the NCS
restraints were removed, and further cycles of manual rebuilding
and positional and temperature factor refinement were necessary to
reduce the crystallographic R-factor and R-free values (in the 20.00-
Gros, P.; Grosse-Kunstleve, R. W.; Jiang, J. S.; Kuszewski, J.;
Nilges, M.; Pannu, N. S.; Read, R. J.; Rice, L. M.; Simonson, T.;
Warren, G. L. Acta Crystallogr Sect D 1998, 54, 905–921.
24. Nicholls, A.; Sharp, K. A.; Honig, B. Proteins 1991, 11, 281–296.
25. Feller, G.; Arpigny, J. L.; Nminx, E.; Geday, C. Camp Biochem
Physiol 1997, 118A, 495–499.
˚
to 2.20-A resolution range) to 16.1 and 20.5%, respectively. Data
refinement statistics are summarized in Table I. Coordinates and
structure factors were deposited in the Protein Data Bank (accession
code 3LS2).
26. Siddiqui, K. S.; Cavicchioli, R. Annu Rev Biochem 2006, 75,
403–433.
27. Saunders, N. F.; Thomas, T.; Curmi, P. M.; Mattick, J. S.; Kuc-
zek, E.; Slade, R.; Davis, J.; Franzmann, P. D.; Boone, D.; Ruster-
holtz, K.; Feldman, R.; Gates, C.; Bench, S.; Sowers, K.; Kadner,
K.; Aerts, A.; Dehal, P.; Detter, C.; Glavina, T.; Lucas, S.;
Richardson, P.; Larimer, F.; Hauser, L.; Land, M.; Cavicchioli, R.
Genome Res 2003, 13, 1580–1588.
REFERENCES
1. Harms, N.; Ras, J.; Reijnders, W. N.; van Spanning, R. J.; Stout-
hamer, A. H. J Bacteriol 1996, 178, 6296–6299.
2. Uotila, L.; Koivusalo, M. J Biol Chem 1974, 249, 7664–7672.
Biopolymers