Three-dimensional structure of binunctional anhydrolase
Andrea Štěpánková1, 2, Tereza Skálová1, Jarmila Dušková1, Tomáš Kovaľ3, Jindřich Hašek1, L. H. Østergaard4, Jan Dohnálek1
1Institute of Macromolecular Chemistry AS CR, v.v.i., Heyrovského nám. 2, 162 06, Prague 6, Czech Republic
2Dept. of Solid State Physics, FNSPE, CTU, Trojanova 13, 120 00, Prague 2, Czech Republic
3Institute of Physic AS CR, Cukrovarnická 10, P162 00, Prague 6, Czech Republic
4 Novozymes A/S, Brudelysvej 26,
DK-2880 Bagsvaerd, Denmarks
Organophosphorus acid anhydrolases (OPAA; E.C. 18.104.22.168) belong to a class of enzymes that are able to hydrolyze a variety of toxic organophosphorus compounds such as pesticides and also some types of chemical nerve agents which are acetylcholinesterase inhibitors. Due to their significant proteolytic activity against various Xaa-Pro dipeptides (Xaa stands for any amino acids), OPAA enzymes also belong to prolidases.
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 at 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.
This work was supported by the Czech Science Foundation projects 310/09/1407 and 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.