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Characterization of recombinant thiamine diphosphate-dependent phosphonopyruvate decarboxylase fromStreptomyces viridochromogenesTü494

Journal of Molecular Catalysis B Enzymatic
Publication Date
DOI: 10.1016/j.molcatb.2009.03.016
  • Thiamine Diphosphate
  • Phosphonopyruvate
  • Decarboxylase
  • Site-Directed Mutagenesis
  • Thiamine Binding Site
  • Biology
  • Chemistry
  • Medicine


Abstract Phosphonopyruvate decarboxylase (PPDC) catalyzes the thiamine diphosphate-dependent non-oxidative decarboxylation of phosphonopyruvate to phosphonoacetaldehyde and carbon dioxide. The enzyme of S. viridochromogenes Tü494 was expressed as a recombinant fusion protein with an N-terminal 10× histidine-tag in Escherichia coli cells, and was purified to homogeneity by nickel affinity chromatography. The biochemical properties of the recombinant enzyme were characterized, measuring phosphonoacetaldehyde formation by two newly developed coupled enzyme assays. PPDC has a high affinity to its cofactors ThDP, and Mg 2+ (both K m ∼ 40 μM). The metal ions Ca 2+ and Mn 2+ ( K m ∼ 3 μM) could substitute for Mg 2+. In coupled enzyme assays at pH 8.0 (HEPES buffer) and at 30 °C, PPDC followed Michaelis–Menten kinetics with phosphonopyruvate, with a K m value of 3.2 ± 0.35 μM and a v max value of 0.81 ± 0.01 U/mg. Neither pyruvate, β-hydroxypyruvate, nor fluoropyruvate served as alternative substrates. Gel filtration chromatography indicated a molecular mass of 72,100 ± 220 Da. Taking into account a subunit size of about 43,600 Da, the quaternary structure of the Ppd appears to be homodimeric. A comparison with the pyruvate decarboxylase (PDC) from Zymomonas mobilis allowed the identification of several amino acid residues, whose potential functions were examined by site-directed mutagenesis. Based on kinetic data of various site-directed PPDC variants and by comparison to 3D structures of PDC and benzoylformate decarboxylase, a model of the active site was generated. As in most other ThDP-dependent enzymes a glutamate residue (Glu-48 of PPDC) appears to be responsible for ThDP activation. Residues Ser-25, His-110, and Asp-297 affect the catalytic activity and are probably located in the direct vicinity of the substrate binding site. Residues Asp-265, Asn-293, and Gly-294 were found within the conserved ThDP-binding motif and mark the binding site of the Me 2+ ion that is responsible for ThDP anchoring. Asp-263 is conserved in PPDC sequences and in sulfopyruvate decarboxylase, and appears to contribute to the metal ion binding site, too. Glu-224, another conserved residue is essential for catalytic activity. The results for Glu-224 and Asp-263 are contrary to the description of the corresponding residues in the PPDC of the gram-negative bacterium Bacteroides fragilis [G. Zhang, J. Dai, Z. Lu, D. Dunaway-Mariano, J. Biol. Chem. 278 (2003) 41302–41308].

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