Structural and functional study of
bi-functional anhydrolase
Andrea Štěpánková1, 2, Tereza
Skálová1, Jarmila Dušková1, Tomáš Kovaľ3, Jindřich
Hašek1, L. H.
Østergaard4, Jan Dohnálek1
1 Institute of Macromolecular Chemistry AS CR, v.v.i., Heyrovského nám. 2, 162 06, Prague 6, Czech Republic
2 Dept. of Solid State Physics, FNSPE, CTU, Trojanova 13, 120 00, Prague 2, Czech Republic
3 Institute of Physic AS CR, Cukrovarnická 10, P162 00, Prague 6, Czech Republic
4 Novozymes A/S, Brudelysvej
26, DK-2880 Bagsvaerd, Denmark
The bacterial enzyme Organophosphorus acid anhydrolase (OPAA) is able to catalyze the hydrolysis of both proline dipeptides (Xaa-Pro) and several types of organophosphate (OP) compounds. The OP compounds are highly toxic they are commonly used as pesticides and unfortunately also as nerve agents. Enzymatic biodegradation is safer and more economical than detoxification via purely chemical means or incineration, so recombinant OPAA can be applied as an enzymatic tool for detoxification of widespread pesticide waste and for deactivation of chemical warfare agents.
Three X-ray structures of recombinant wild
type enzyme OPAA from a marine bacterium Alteromonas macleodii are
presented here. The data were collected at the beam line BL14.1 of the source
of synchrotron radiation Bessy II (Helmholtz-Zentrum, Berlin). In two cases,
the crystals belong to space group C2 with unit cell parameters a
= 134.3 Å, b =
49.1 Å, c =
97.2 Å and β = 125.0°. Data were
collected up to resolutions 1.8 Å and 1.9 Å, respectively. In other
case, the crystal belongs to space group P212121
with unit cell parameters a = 75.6 Å, b = 111.2 Å, c
= 138.1 Å and data were collected to the resolution 2.2 Å.
The data were processed using HKL2000 and the structures were refined by Refmac5. All the 442 amino acids of the recombinant protein were located in electron density. The protein fold is mainly α-helical. The binuclear metal center is located within the pita bread domain in the active site. Manganese ions, which are required for protein activity, were observed in full occupancy in the active sites. The enzyme form dimers. Existence of dimers was confirmed both in crystal and also in solution by dynamic light-scattering. The enzyme shares the so-called “pita bread” fold of the C-terminal domain with other enzymes with prolidase activity.
1 Vyas,
N. K. et al., (2010). Structural insights into the
dual activities of the nerve agent degrading organophosphate
anhydrolase/prolidase.
Biochemistry, 49, 547-559.
2. Raushel,
F. M. (2002). Bacterial detoxification of organophosphate
nerve agents. Current opinion in Microbiology,
5, 288-295.
Acknowledgment: This work was supported by the Czech Science Foundation project 305/07/1073. The authors wish to thank Dr. Uwe Müller of the Helmholtz-Zentrum Berlin, Albert-Einstein-Str. 15 for support at the beam line BL14.1 of Bessy II.