title: Evidence for a dual role of an active site histidine in α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase.
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.
authors: Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, P.O. Box 4098, Atlanta, GA 30303, USA.
authors: Lu
authors: L
authors: Huo
authors: Andrew J
authors: AJ
authors: Fielding
authors: Yan
authors: Y
authors: Chen
authors: Tingfeng
authors: T
authors: Li
authors: Hiroaki
authors: H
authors: Iwaki
authors: Jonathan P
authors: JP
authors: Hosler
authors: Lirong
authors: L
authors: Chen
authors: Yoshie
authors: Y
authors: Hasegawa
authors: Lawrence
authors: L
authors: Que
authors: Aimin
authors: A
authors: Liu
mesh_terms: D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004295:Dihydroxyphenylalanine / Q000378:metabolism; D005290:Ferric Compounds / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011551:Pseudomonas fluorescens / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity
chemical_list: D005290:Ferric Compounds; D008670:Metals; D006639:Histidine; D004295:Dihydroxyphenylalanine; D002262:Carboxy-Lyases; C024396:aminocarboxymuconate-semialdehyde decarboxylase