Kinetic and Spectroscopic Characterization of ACMSD fromPseudomonas fluorescensReveals a Pentacoordinate Mononuclear Metallocofactor

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Abstract
The enzyme α-amino-β-carboxy-muconic-ε-semialdehyde decarboxylase (ACMSD) plays an important role in the biodegradation of 2-nitrobenzoic acid in microorganisms and in tryptophan catabolism in humans. We report that the overexpressed ACMSD enzyme from Pseudomonas fluorescens requires a divalent metal, such as Co(II), Fe(II), Cd(II), or Mn(II), for catalytic activity and that neither a redox reagent nor an organic cofactor is required for the catalytic function. The metal ions can be taken up in either cell or cell-free preparations for generating the active form of ACMSD. The kinetic parameters and enzyme specific activity are shown to depend on the metal ion present in the enzyme, suggesting a catalytic role of the metal center. EPR spectrum of Co(II)-ACMSD provides a high-spin (S = 3/2) mononuclear metal ion in a non-heme, noncorrinoid environment with a mixed nitrogen/oxygen ligand field. We observe hyperfine interactions due to 59Co nucleus at temperatures below 5 K but not at higher temperatures. Ten hyperfine lines are present in the g⊥ region, and three equivalent nitrogen hyperfine couplings are required to simulate the resonances in the EPR spectrum. The results for the metal binding site are also assessed using the copper-substituted enzyme, and the EPR spectral assignments for both cobalt and copper proteins give strong support for a distorted trigonal bipyramidal geometry of the metal center. Ultimately, these results suggest for the first time that ACMSD is a metal-dependent enzyme that catalyzes a novel nonoxidative decarboxylation.

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