| Abstract
| - The enzymatic activity of Pseudomonas fluorescens α-amino-β-carboxymuconic-ε-semialdehydedecarboxylase (ACMSD) is critically dependent on a transition metal ion [Li, T., Walker, A. L., Iwaki,H., Hasegawa, Y., and Liu, A. (2005) J. Am. Chem. Soc. 127, 12282−12290]. Sequence analysis in thisstudy further suggests that ACMSD belongs to the amidohydrolase superfamily, whose structurallycharacterized members comprise a catalytically essential metal cofactor. To identify ACMSD's metalligands and assess their functions in catalysis, a site-directed mutagenesis analysis was conducted. Alterationof His-9, His-177, and Asp-294 resulted in a dramatic loss of enzyme activity, substantial reduction ofthe metal-binding ability, and an altered metallocenter electronic structure. Thus, these residues areconfirmed to be the endogenous metal ligands. His-11 is implicated in metal binding because of thestrictly conserved HxH motif with His-9. Mutations at the 228 site yielded nearly inactive enzyme variantsH228A and H228E. The two His-228 mutant proteins, however, exhibited full metal-binding ability anda metal center similar to that of the wild-type enzyme as shown by EPR spectroscopy. Kinetic analysison the mutants indicates that His-228 is a critical catalytic residue along with the metal cofactor. Sincethe identified metal ligands and His-228 are present in all known ACMSD sequences, it is likely thatACMSD proteins from other organisms contain the same cofactor and share similar catalytic mechanisms.ACMSD is therefore the first characterized member in the amidohydrolase superfamily that represents aC−C breaking activity.
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