The phosphonopyruvate decarboxylase from Bacteroides fragilis.
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.
Zhang
Guofeng
G
Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131-0001, USA.
Dai
Jiaying
J
Lu
Zhibing
Z
Dunaway-Mariano
Debra
D
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001441:Bacteroides fragilis / Q000201:enzymology*; D002240:Carbohydrate Sequence; D002262:Carboxy-Lyases / Q000737:chemistry*; D002384:Catalysis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011134:Polysaccharides / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001426:Bacterial Proteins; D007477:Ions; D011134:Polysaccharides; D002262:Carboxy-Lyases; C113679:PPD protein, Streptomyces viridochromogenes