Domain of unknown function (DUF) families constitute 3,892 of the 16,295 families in the Pfam database (release 29.0). Given their biological importance, large-scale strategies are required to accomplish their functional assignments. Here, we illustrate an integrated “genomic enzymology” strategy to identify diverse functions within the DUF1537 family (PF07005). We combined high-throughput ligand screening results for transport system solute binding proteins with the synergetic analysis of sequence similarity networks and genome neighborhood networks to establish that the members of the DUF1537 family are novel ATP-dependent four-carbon sugar kinases. This study illustrates the utility of this strategy and enhances our knowledge of bacterial carbohydrate catabolism.