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)(OH2) active site. The water ligand is H-bonded to a conserved His228 residue adjacent to the metal center in ACMSD from Pseudomonas fluorescences (PfACMSD). Site directed mutagenesis of His228 to tyrosine and glycine in the present study results in 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 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 (LMCT). Co(II)-substituted H228Y ACMSD is brown in color and exhibits an EPR spectrum showing a high-spin Co(II) center with a well-resolved 59Co (I = 7/2) eight-line hyperfine splitting pattern. The X-ray crystal structures of the as-isolated Fe-H228Y (2.8 Å), Co- (2.4 Å) and Znsubstituted 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 solved. 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 His228 mutation. Together, these results highlight the importance of His228 for PfACMSD’s metal specificity as well as maintaining a water molecule as 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.

Functional Characterization of a Novel Member of the Amidohydrolase 2 Protein Family, 2-Hydroxy-1-Naphthoic Acid Nonoxidative Decarboxylase from Burkholderia sp. Strain BC1

“…Interestingly, substitution of another conserved histidine residue (His204), not directly involved in metal binding, leads to the complete inactivation of the protein. This result is similar to that observed in ACMSD, where the corresponding residue (His228) was suggested to play the role of an acid-base catalyst involved in deprotonation of the metal-bound water, facilitating the decarboxylation of ACMS (47). On the other hand, docking analysis revealed that carboxyl and hydroxyl groups of 2H1NA are hydrogen bonded with Arg33/Tyr272 and Tyr272 of HndA, respectively.…”

The Power of Two

“…Protein Preparation, SDS-PAGE, and Size Exclusion Chromatography-Expression and purification of ACMSD proteins followed the protocol established previously (10,11,22,24) with a few minor changes. Rather than using pH 8.0 buffer during affinity chromatography purification, buffer at pH 7.4 was used to increase protein stability of the mutants.…”

“…Enzyme Activity Assay-The substrate ACMS was generated enzymatically by catalyzing the dioxygenation of 3-hydroxyanthranilic acid in oxygen saturated buffer with ferrous 3-hydroxyanthranilate 3,4-dioxygenase containing no free transient metal ion as reported previously (10,11,22). Briefly, the ACMSD enzyme assay mixture contained 5-120 M ACMS and an appropriate amount of ACMSD protein in 25 mM HEPES buffer with 5% glycerol, pH 7.0.…”

“…All single crystals were obtained following the crystallization conditions described in the previous studies (19,22). For the heterodimer crystal, R51A and R239A were mixed for more than 48 h at 4°C before being used for crystallization by hanging drop vapor diffusion in VDX plates (Hampton Research).…”

“…In fact, it is still unclear whether the catalytically active form of human ACMSD in solution is a monomeric or dimeric enzyme at the present time. All of the P. fluorescens ACMSD structures determined thus far, including wild type and mutants charged with different transition metals, are dimeric (19,22). If the monomeric form is catalytically active, then only one conserved arginine residue, Arg-51, is present in the active site of the enzyme in both human ACMSD and P. fluorescens ACMSD structures.…”