4ERA

Evidence for a Dual Role of an Active Site Histidine in alpha-Amino-beta-Carboxymuconate-epsilon-Semialdehyde Decarboxylase

Summary for 4ERA

• Entry DOI: 10.2210/pdb4era/pdb
• Related: 4EPK 4ERG 4ERI
• Descriptor: 2-amino-3-carboxymuconate 6-semialdehyde decarboxylase, COBALT (II) ION (3 entities in total)
• Functional Keywords: tim-barrel, decarboxylase, metal-binding, co(ii), lyase
• Biological source: Pseudomonas fluorescens
• Total number of polymer chains: 2
• Total formula weight: 74535.11

Authors

Huo, L.,Fielding, A.J.,Chen, Y.,Li, T.,Iwaki, H.,Hosler, J.P.,Chen, L.,Hasegawa, Y.,Que Jr., L.,Liu, A. (deposition date: 2012-04-19, release date: 2012-08-22, Last modification date: 2023-09-13)

Primary citation

Huo, L.,Fielding, A.J.,Chen, Y.,Li, T.,Iwaki, H.,Hosler, J.P.,Chen, L.,Hasegawa, Y.,Que, L.,Liu, A.
Evidence for a Dual Role of an Active Site Histidine in alpha-Amino-beta-Carboxymuconate-epsilon-Semialdehyde Decarboxylase
Biochemistry, 51:5811-5821, 2012

PubMed Abstract: The previously reported crystal structures of α-amino-β-carboxymuconate-ε-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 Å) and Co-substituted (2.4 Å) and Zn-substituted H228Y (2.0 Å resolution) support the spectroscopic assignment of metal ligation of the Tyr228 residue. The crystal structure of Zn-H228G (2.6 Å) 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.

PubMed: 22746257
DOI: 10.1021/bi300635b

Experimental method

X-RAY DIFFRACTION (2.398 Å)