A gene encoding for arabinose 5-phosphate isomerase (API), which catalyzes the interconversion of d-ribulose 5-phosphate (Ru5P) and d-arabinose 5-phosphate (A5P), has been identified from the genome of Escherichia coli K-12. API is the first enzyme in the biosynthesis of 3-deoxy-d-manno-octulosonate (KDO), a sugar moiety located in the lipopolysaccharide layer of most Gram-negative bacteria. The API gene yrbH is located next to the recently identified specific KDO 8-P phosphatase gene, yrbI. The 328-amino acid open reading frame yrbH was cloned, overexpressed, and characterized. The purified recombinant enzyme is a tetramer and is sensitive to inhibition by zinc cations. API has optimal activity at pH 8.4 and catalytic residues with estimated pKa values of 6.55 +/- 0.04 and 10.34 +/- 0.07. The enzyme is specific for A5P and Ru5P, with apparent Km values of 0.61 +/- 0.06 mm for A5P and 0.35 +/- 0.08 mm for Ru5P. The apparent kcat in the A5P to Ru5P direction is 157 +/- 4 s-1, and in the Ru5P to A5P direction it is 255 +/- 16 s-1. The value of Keq (Ru5P/A5P) is 0.50 +/- 0.06. Homology searches of the E. coli genome suggest yrbH may be one of multiple genes that encode proteins with API activity.