Abstract
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 (kcat = 10.2 +/- 0.3 s-1) the decarboxylation of phosphonopyruvate (Km = 3.2 +/- 0.2 microm) to phosphonoacetaldehyde (Ki = 15 +/- 2 microm) and carbon dioxide at an optimal pH range of 7.0-7.5. Thiamine pyrophosphate (Km = 13 +/- 2 microm) and certain divalent metal ions (Mg(II) Km = 82 +/- 8 microm; Mn(II) Km = 13 +/- 1 microm; Ca(II) Km = 78 +/- 6 microm) serve as cofactors. Phosphonopyruvate decarboxylase is a member of the alpha-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 alpha-ketodecarboxylase family, was shown by site-directed mutagenesis to play an important role in catalysis. Pyruvate (kcat = 0.05 s-1, Km = 25 mm) and sulfopyruvate (kcat approximately 0.05 s-1; Ki = 200 +/- 20 microm) are slow substrates for the phosphonopyruvate decarboxylase, indicating that this enzyme is promiscuous.
Highlights
Bacteroides fragilis is a human pathogen that causes intraabdominal abscess formation in its host [1, 2]
The AEP pathway enzymes of B. fragilis have not been isolated for characterization
The recombinant Ppyr decarboxylase was purified to homogeneity by using the 4-step protocol summarized in Table I in an overall yield of 3.7 mg/g wet cells
Summary
Bacteroides fragilis is a human pathogen that causes intraabdominal abscess formation in its host [1, 2]. AepX, aepY, and aepZ, which encode proteins that share significant sequence identity with the three enzymes of the AEP biosynthetic pathway, are included within this locus (Fig. 2). What we do know about this enzyme derives from the study of the bacterial enzyme which functions in biosynthetic pathways leading to bialaphos, fosfomycin, and phosphinothricin tripeptide in Streptomyces hygroscopicus [22], Streptomyces wendmorensis [23, 24], and Streptomyces viridomogenes [25], respectively In these pathways, P-enolpyruvate mutase and Ppyr decarboxylase collaborate to form the common precursor phosphonoacetaldehyde (Pald). As with the Ppyr decarboxylase of Tetrahymena pyriformis, the third enzyme of the pathway, Pald transaminase proved to be membrane-bound and difficult to isolate. We report the cloning and overexpression of the B. fragilis Ppyr decarboxylase gene (aepY), purification of the protein, and for the first time, an in-depth study of the Ppyr decarboxylase
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