Dihydroxyacetone kinases are a family of sequencerelated enzymes that utilize either ATP or a protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) as a source of high energy phosphate. The PTS is a multicomponent system involved in carbohydrate uptake and control of carbon metabolism in bacteria. Phylogenetic analysis suggests that the PTS-dependent dihydroxyacetone kinases evolved from an ATPdependent ancestor. Their nucleotide binding subunit, an eight-helix barrel of

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... ar up-down topology, retains ADP as phosphorylation site for the double displacement of phosphate from a phospho-histidine of the PTS protein to dihydroxyacetone. ADP is bound essentially irreversibly with a t1 ⁄2 of 100 min. Complexation with ADP increases the thermal unfolding temperature of dihydroxyacetone L from 40 (apo-form) to 65°C (holoenzyme). ADP assumes the same role as histidines, cysteines, and aspartic acids in histidine kinases and PTS proteins. This conversion of a substrate binding site into a cofactor binding site reflects a remarkable instance of parsimonious evolution.