Abstract
The crystal structure of N-carbamoyl-D-amino-acid amidohydrolase (D-NCAase) from Agrobacterium radiobacter, an industrial biocatalyst for the production of valuable D-amino acids, has been determined to a resolution of 1.95 Å. The active site residue Cys172 has been proposed as the residue involved in a nucleophilic attack of the C atom of the carbamoyl group in a substrate. Docking simulation was performed to predict the substrate-binding pocket by using Grid-docking method. Based on the docking model, the Cys172 was substituted with Ser or Ala to inactivate the enzyme but did not abolish the ability to bind the substrate. Both mutant enzymes, Cys172Ser and Cys172Ala have been crystallized and co-crystallized with the substrate, N-carbamoyl-D-p-hydroxyphenylglycine (D-NCHPG). The crystal structures of Cys172Ala and Cys172Ser were determined respectively in its native form and in complex form with N-carbamoyl-D-p-hydroxyphenylglycine. Analysis of these structures suggest that (i) the overall protein folding, side chain conformations and active sites of both mutants, mutant-substrate complexes are isomorphous with those of wild-type enzyme, (ii) the mutated side chains are pointed for catalysis in a similar conformation as that observed for the wild-type D-NCAase, (iii) within the identified substrate binding pockets, a number of residues including Asn173. Arg175, Arg176, Glu47, Lys127, His144 and Glu146 are involved in interacting with the substrate, and (iv) the role of Cys172 in enzymatic activity and the binding mode of the substrate were further confirmed in the crystal structures. Residues Asn173, Arg175, and Arg176 nearby the carboxyl moiety of the substrate were investigated by site-directed mutagenesis. Crystal structures of Arg175Ala and Asn173Ala D-NCAase were obtained. Structural comparisons of wild type D-NCAase and R175A crystal structures reveal significant conformational changes in the active site. No conformational change is found in the active site of N173A crystal structure. Arg175 and Arg176 were further mutated to Lys residues respectively. Kinetics analysis shows no detectable activity of the Arg175Ala and Arg176Ala mutants and increase Km values of the Arg175Lys and Arg176Lys mutants while a decrease in Km value for Asn173Ala mutant. Site-directed mutagenesis results are discussed in light of the crystal structures of mutant D-NCAase and mutant-substrate complexes.