The two consecutive activities of the cobalamin biosynthetic pathway that catalyze the conversion of cobinamide to cobinamide phosphate (cobinamide kinase) and of cobinamide phosphate to GDP-cobinamide (cobinamide phosphate guanylytransferase) were shown to be carried by the same protein in Pseudomonas denitrificans. This bifunctional protein was purified to homogeneity by high-performance liquid chromatography of extracts of a recombinant strain of this microorganism, and the sequence of the first 10 amino acid residues at the N terminus was determined. Both activities were specific to the coenzyme forms of the corrinoid substrates and exhibited an optimum pH at 8.8. Both ATP and GTP were shown to be in vitro gamma-phosphate donors for cobinamide kinase. However, competition experiments demonstrated that ATP was the preferred substrate, a result that can be explained in terms of the kinetic properties of the enzyme. Labeling experiments established that the phosphate group of cobinamide phosphate is quantitatively retained as the inner phosphate of GDP-cobinamide during the guanylyltransferase reaction. The native protein had an apparent molecular weight of 40,000, as estimated by gel filtration, and consisted of two identical subunits of Mr 20,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein had an isoelectric point of 5.35 and contained a high-affinity GTP-binding site (Kaff.(GTP) = 0.22 microM). Binding of GTP onto this site resulted in a marked increase of the affinity of cobinamide kinase for cobinamide. This property and other kinetic properties may regulate the enzyme and prevent the accumulation of cobinamide phosphate.