The structural and biochemical data of enzyme capable of organophosphates degradation

 

Andrea Štěpánková1, J. Dušková2, T. Skálová2, J. Hašek2, T. Kovaľ3, L. H.  Østergaard4,  J. Dohnálek2

 

1  Dept. of Solid State Physics, FNSPE, CTU, Trojanova 13, 120 00, Prague 2, Czech Republic

2   Institute of Macromolecular Chemistry AS CR, v.v.i., Heyrovského nám. 2, 162 06, Prague 6, Czech Republic

3 Institute of Physic AS CR, Cukrovarnická 10, 162 00, Prague 6, Czech Republic

4  Novozymes A/S, Brudelysvej 26, DK-2880 Bagsvaerd, Denmark

a.stepanko@gmail.com

 

Extremophiles are organisms living in extreme conditions on Earth (for illustration: temperatures of -50 °C or 113 °C, hydrostatic pressures of 120 MPa or pH values of 0.5 or 12.0). Psychrotrophs are a group of extremophilic microorganisms with the minimal temperature of growth around 0 °C and with the optimal temperature of growth around 20 °C. Halophiles are a group of extremophilic microorganisms requiring at least 0.2 M concentrations of salt for their growth.

The examined enzyme organophosphorus acid anhydrolase (OPAA) is able to catalyze hydrolysis of proline dipeptides (Xaa-Pro), and of several types of organophosphate compounds commonly used as pesticides or as nerve agents. The enzyme, with the pH optimum around 8, offers a large potential for biotechnological application as a tool for bio-degradation of these dangerous compounds. Two different types of the OPAA enzyme from different bacteria – psychrothropic and slightly halophilic Pseudoalteromonas haloplanktis and slightly halophilic Alteromonas macleodii – were studied and compared with the  sequence related human prolidase.

Three molecular structures of the OPAA enzyme from Alteromonas macleodii have been determined. The structure data were collected at the beam line PX 14.1 of the source of synchrotron radiation Bessy II (Helmholtz-Zentrum, Berlin). Native amOPAA crystallized in the space group C2 with unit cell parameters a = 134.3 Å, b = 49.1 Å, c = 97.2 Å and β = 125.0°. The crystals were measured native and soaked with ligand Pro-Gly, too. Data were collected up to resolutions 1.8 Å and 1.9 Å, respectively. The third crystal structure determined is the native amOPAA crystallized in the space group P212121 (unit cell parameters a = 75.6 Å, b = 111.2 Å, c = 138.1 Å). Data were collected to resolution 2.2 Å in this case. Refinement of all these structures performed well with the average B factor around 20 Å2 and the final R factors in the range 15.3 – 16.6 %. The structure of was deposited in the PDB under the accession code 3RVA.

opaa-MT2.png

 

Fig. 1. The cartoon representation of 3D structure of  the OPAA from Alteromonas macleodii. The binuclear metal center is highlighted by two spheres.

 

To modulate the enzymatic activity profiles and to explain the enzymatic function of OPAA, we made site-directed mutagenesis of OPAA from Pseudoalteromonas haloplanktis near in its substrate binding site (Tyr212, His226, Asp244, Asp255, His335, His339, Arg370, Glu384, Arg421, Glu423, Val345, His346) found in our structure with the dipeptide Pro-Gly complex. Only two of seven tested single-mutations (Y212F, Y212S - H226N, H226K - H334N, H334K, H334Q) retained the enzymatic activity.

Mutations.jpg

Fig. 2. Tested mutations around the active site of OPAA. The red residus are in positions found in the native OPAA. The magenta residues are residues which were mutated in phOPAA; the green residues show the particular mutations. The manganese ions are shown as green spheres.

 

Enzyme assay studies showed a loss of activity as a consequence of site-directed mutagenesis in five cases. Two mutations highlighted in bold retained the enzymatic activity according to the results of biochemical characterization (DLS, SDS-PAGE). The pH profiles of the OPAA mutants Y212F and H226K preserving activity remain similar to the wild type, but in the case of mutants are shifted slightly to more basic pH.

The comparative study shows that the OPAA enzyme is very similar to human prolidase. High similarities in structure and also in substrate specificities of OPAAs and prolidases have brought up some interesting questions regarding the historical classification of this group of enzymes with related but not identical activity profiles.

The structures discussed here together with other three structures of protein-ligand complexes of the low temperature active β-galactosidase are part of the PhD thesis /3/.

References:

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.

3.       Štěpánková A., (2012). PhD Thesis, X-ray structural analysis of enzymes from extremophiles and their complexes with bound ligands, pp. 1-119, FJFI ČVUT, Praha.