X-RAY CRYSTALLOGRAPHY OF RECOMBINANT LACTOCCOCUS LACTIS PROLIDASE

Abstract

Prolidase has potential applications in cheese debittering, organophosphate detoxification and as an enzyme replacement therapy in prolidase-deficient patients. Recombinant Lactococcus lactis prolidases and their catalytic properties have previously been characterized in Dr. Tanaka's research group. Unlike other prolidases, L. lactis prolidase shows allosteric behaviour, metal-dependent substrate specificity and substrate inhibition. The current project focuses on elucidating the three-dimensional structure of L. lactis prolidase using X-ray crystallography. Hexagonal plate-like crystals of wild-type L. lactis prolidase were grown by the hanging drop vapour diffusion method, allowing the crystals to grow to about 50 µm in their longest dimension. The crystallization cocktail in which they grew contained 0.08 M sodium cacodylate (pH 6.5), 0.16 M calcium acetate, 14 % PEG 8000 and 18 % glycerol. Crystal diffraction data was collected at a wavelength of 0.9795 Å on beamline 08ID-1 of the Canadian Macromolecular Crystallography Facility at the Canadian Light Source and was processed using X-ray Detector Software. The crystals belonged to space group C2 and estimated to contain three molecules in an asymmetric unit. The electron density map of this structure was solved by the molecular replacement method and the structure model was refined against 2.25 Å resolution data. Molecule A forms a dimer with molecule B, while molecule C forms a dimer with molecule C', which is located in the neighbouring crystal asymmetric unit. The electron density of molecule A was well-defined and complete. Therefore, all the 362 amino acid residues of L. lactis prolidase were fitted. The other two molecules were incomplete and less defined. Only 360 and 352 residues could be fitted in molecules B and C, respectively. Molecule C, the worst of the three, compromised the overall quality of the refined structure. However, the functional interpretation of the structure was not compromised since the well-defined molecules form a dimer with each other and the biologically-functional form of L. lactis prolidase is a homodimer. The final Rwork and Rfree are 22.39 and 27.77, respectively. Comparison with other known prolidases revealed that Asp 36 and His 38 are unique to L. lactis prolidase. These residues have been shown to be involved in the allosteric behaviour and substrate inhibition of this enzyme, respectively. Therefore, this crystal structure further supports their suggested contribution in L. lactis prolidase's unique catalytic properties.