Escherichia coli YrbI is 3-deoxy-D-manno-octulosonate 8-phosphate phosphatase

Abstract

3-Deoxy-D-manno-octulosonate 8-phosphate (KDO 8-P) phosphatase, which catalyzes the hydrolysis of KDO 8-P to KDO and inorganic phosphate, is the last enzyme in the KDO biosynthetic pathway for which the gene has not been identified. Wild-type KDO 8-P phosphatase was purified from Escherichia coli B, and the N-terminal amino acid sequence matched a hypothetical protein encoded by the E. coli open reading frame, yrbI. The yrbI gene, which encodes for a protein of 188 amino acids, was cloned, and the gene product was overexpressed in E. coli. The recombinant enzyme is a tetramer and requires a divalent metal cofactor for activity. Optimal enzymatic activity is observed at pH 5.5. The enzyme is highly specific for KDO 8-P with an apparent Km of 75 μm and a kcat of 175 s-1 in the presence of 1 mm Mg2+. Amino acid sequence analysis indicates that KDO 8-P phosphatase is a member of the haloacid dehalogenase hydrolase superfamily.