4EPK: Evidence For A Dual Role Of An Active Site Histidine In Alpha-Amino- Beta-Carboxymuconate-Epsilon-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)(OH(2)) 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 (59)Co (I = (7)/(2)) eight-line hyperfine splitting pattern. The X-ray crystal structures of as-isolated Fe-H228Y (2.8 A) and Co-substituted (2.4 A) and Zn-substituted H228Y (2.0 A resolution) support the spectroscopic assignment of metal ligation of the Tyr228 residue. The crystal structure of Zn-H228G (2.6 A) 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.
PDB ID: 4EPKDownload
MMDB ID: 102176
PDB Deposition Date: 2012/4/17
Updated in MMDB: 2012/08
Experimental Method:
x-ray diffraction
Resolution: 2.6  Å
Source Organism:
Similar Structures:
Biological Unit for 4EPK: dimeric; determined by author and by software (PISA)
Molecular Components in 4EPK
Label Count Molecule
Proteins (2 molecules)
2-amino-3-carboxymuconate 6-semialdehyde Decarboxylase
Molecule annotation
Chemicals (3 molecules)
* Click molecule labels to explore molecular sequence information.

Citing MMDB