Evidence for a Dual Role of an Active Site Histidine in α-Amino-β-carboxymuconate-ε-semialdehyde Decarboxylase

The previously reported crystal structures of alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) show a five-coordinate Zn(II)(His)(3)(Asp)(OH2) active site. The water ligand is H-bonded to a conserved His228 residue adjacent to the metal center in ACMSD from Pseudomonas fluorescens (PfACMSD). Site-directed mutagenesis of His228 to tyrosine and glycine in this study results in a complete or significant loss of activity. Metal analysis shows, that H228Y and H228G contain iron rather than zinc, indicating that this residue plays a role in the metal selectivity of the protein. As isolated H228Y displays a blue color, which is not seen in wild type ACMSD. Quinone staining and resonance Raman analyses indicate that the blue color originates from Fe(III)-tyrosinate ligand-to-metal charge transfer Co(II)-substituted H228Y ACMSD is brown in color and exhibits an electron paramagnetic resonance spectrum showing a high-spin Co(II) center with a well resolved Co-59 (I = 7/2) eight line hyperfine splitting pattern The X-ray crystal structures Of as isolated Fe-H228Y (2.8 angstrom) and Co-substituted (2.4 angstrom) and Zn-substituted H228Y (2.0 angstrom resolution) support the spectroscopic assignment of Metal ligation of the Tyr228 residue. The crystal structure of Zn H228G (2.6 angstrom) was also determined. These four structures show that the water ligand present in WT Zn-ACMSD is either missing (Fe-H228Y, Co-H228Y, and Zn-H228G). or disrupted. (Zn-H228Y) in response to the His228 mutation. Together, these results highlight the importance of His228 for PfACMSD's Metal specificity as well as maintaining a water molecule as a ligand of the metal center. His228 is thus proposed to play a role in activating the metal-bound water ligand for subsequent nucleophilic attack on the substrate.