Conservation of function across families of orthologous enzymes is generally accompanied by conservation of their active site electrostatic potentials. To study the electrostatic conservation in the highly conserved essential enzyme, thymidylate synthase (TS), we conducted a systematic species-based comparison of the electrostatic potential in the vicinity of its active site. Whereas the electrostatics of the active site of TS are generally well conserved, the TSs from minimal organisms do not conform to the overall trend. Since the genomes of minimal organisms have a high thymidine content compared to other organisms, the observation of non-conserved electrostatics was surprising. Analysis of the symbiotic relationship between minimal organisms and their hosts, and the genetic completeness of the thymidine synthesis pathway suggested that TS from the minimal organism Wigglesworthia glossinidia (W.g.b.) must be active. Four residues in the vicinity of the active site of Escherichia coli TS were mutated individually and simultaneously to mimic the electrostatics of W.g.b TS. The measured activities of the E. coli TS mutants imply that conservation of electrostatics in the region of the active site is important for the activity of TS, and suggest that the W.g.b. TS has the minimal activity necessary to support replication of its reduced genome. The electrostatic potential of a protein plays a crucial role in steering ligands to their binding sites, and orienting them correctly for binding 1. In enzymes, the active site electrostatic potential is important for stabilizing the transition state and thereby catalyzing the reaction 2. Therefore, conservation of protein function across a protein family is often accompanied by conservation of the electrostatic potential in the active site region, even though the rest of the protein may lack a conserved electrostatic potential 3,4. Consequently, comparison of protein electrostatic potentials has been employed as a tool to predict protein function and to derive similarities in protein function across protein families 5–7. Optimizing the electrostatic complementarity between a ligand and the binding site of a protein is also an important aspect in drug design 8,9 and may provide a route to gain target selectivity 10 .