Gas tunnel engineering of prolyl hydroxylase reprograms hypoxia signaling in cells

Molecular engineering of biocatalysts has revolutionized complex synthetic chemistry and sustainable catalysis. Here, we show that it is also possible to use engineered biocatalysts to reprogram signal transduction in human cells. More specifically, we manipulate cellular hypoxia (low O(2)) signaling by engineering the gas-delivery tunnel of prolyl hydroxylase 2 (PHD2), an iron-dependent enzymatic O(2) sensor. Using computational modeling and rational protein design techniques, we resolve PHD2’s gas tunnel and critical residues therein that limit the flow of O(2) to PHD2’s catalytic core. Systematic modification of these residues open the constriction topology of PHD2’s gas tunnel with the most effectively designed mutant displaying 11-fold enhanced hydroxylation efficiency. Furthermore, transfection of plasmids that express these engineered PHD2 mutants in HEK-293T cells reveal significant reduction in the levels of hypoxia inducible factor (HIF-1α) even under hypoxic conditions. Our studies reveal that activated PHD2 mutants can reprogram downstream HIF pathways in cells to simulate physiological O(2)-like conditions despite extreme hypoxia and underscores the potential of engineered biocatalysts in controlling cellular function.