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J. Biol. Chem., Vol. 278, Issue 42, 41302-41308, October 17, 2003

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The Phosphonopyruvate Decarboxylase from Bacteroides fragilis^*

Guofeng Zhang, Jiaying Dai, Zhibing Lu and Debra Dunaway-Mariano 

From the Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131-0001

The Bacteroides fragilis capsular polysaccharide complex is the major virulence factor for abscess formation in human hosts. Polysaccharide B of this complex contains a 2-aminoethylphosphonate functional group. This functional group is synthesized in three steps, one of which is catalyzed by phosphonopyruvate decarboxylase. In this paper, we report the cloning and overexpression of the B. fragilis phosphonopyruvate decarboxylase gene (aepY), purification of the phosphonopyruvate decarboxylase recombinant protein, and the extensive characterization of the reaction that it catalyzes. The homotrimeric (41,184-Da subunit) phosphonopyruvate decarboxylase catalyzes (k_cat = 10.2 ± 0.3 s^–1) the decarboxylation of phosphonopyruvate (K_m = 3.2 ± 0.2 µM) to phosphonoacetaldehyde (K_i = 15 ± 2 µM) and carbon dioxide at an optimal pH range of 7.0–7.5. Thiamine pyrophosphate (K_m = 13 ± 2 µM) and certain divalent metal ions (Mg(II) K_m = 82 ± 8 µM; Mn(II) K_m = 13 ± 1 µM; Ca(II) K_m = 78 ± 6 µM) serve as cofactors. Phosphonopyruvate decarboxylase is a member of the -ketodecarboxylase family that includes sulfopyruvate decarboxylase, acetohydroxy acid synthase/acetolactate synthase, benzoylformate decarboxylase, glyoxylate carboligase, indole pyruvate decarboxylase, pyruvate decarboxylase, the acetyl phosphate-producing pyruvate oxidase, and the acetate-producing pyruvate oxidase. The Mg(II) binding residue Asp-260, which is located within the thiamine pyrophosphate binding motif of the -ketodecarboxylase family, was shown by site-directed mutagenesis to play an important role in catalysis. Pyruvate (k_cat = 0.05 s^–1, K_m = 25 mM) and sulfopyruvate (k_cat 0.05 s^–1; K_i = 200 ± 20 µM) are slow substrates for the phosphonopyruvate decarboxylase, indicating that this enzyme is promiscuous.

Received for publication, June 6, 2003 , and in revised form, August 5, 2003.

^* This work was supported by National Institutes of Health Grants GM 28688 and GM 61099. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom corresponding should be addressed. Tel.: 505-277-3776; Fax: 505-277-6202; E-mail: dd39{at}unm.edu.

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