Evolutionary Motif Swapping of Human Dihydrofolate Reductase Rewires the Enzymatic Cycle

Despite common descent, enzymes often rescue poorly when expressed across domains of life. Causes include factors external to the enzyme, such as differences in codon usage, sensitivity to proteases, and transcriptional or post-translational regulatory differences, yet often the underlying cause remains unclear. Dihydrofolate reductase (DHFR) catalyzes the same metabolic conversion across the tree of life. Nevertheless, human DHFR (hsDHFR) does not effectively rescue growth of DHFR-deficient E. coli despite similar in vitro kinetics. This phenomenon has been previously attributed to inhibition of hsDHFR by its oxidized cofactor, NADP⁺. To understand this phenomenon, we designed mutants based on deep sequence divergences across the tree of life, yielding variants which outperform both wild-type enzymes in vitro and which rescue growth of E. coli. Remarkably, a single, ancient sequence insertion underlies gain of function, not by modulating product inhibition, but by redirecting ligand flux – the non-equilibrium sequence of steps binding and unbinding product, cofactor, and substrate. We find that deleting this insertion decouples the dynamics of a substrate binding loop from subdomain motion, thereby controlling a critical enzymatic parameter orthogonal to catalytic proficiency.