Guiding covalent catalysis enables a carbon-inserting rearrangement in molybdenum cofactor biosynthesis.

Many enzymes catalyze chemically complex rearrangement reactions, yet the molecular strategies that enable precise atomic control often remain enigmatic. One of Nature's most intricate examples occurs in the biosynthesis of the molybdenum cofactor (Moco), a pterin-based cofactor essential to all domains of life. Formation of Moco's characteristic pyranopterin requires a remarkable rearrangement of GTP, in which the C8 atom of the guanine base is inserted between the C-2' and C-3' atoms of ribose. Remarkably, this transformation is catalyzed by a single enzyme, MoaC, yet how MoaC orchestrates this rearrangement remains unclear. Here, we show that MoaC employs an unexpected covalent catalytic mechanism. Using chemical trapping, enzyme kinetics, mass spectrometry, and X-ray crystallography, we identified four kinetically relevant covalent intermediates. The transient covalent linkage forms between Lys131 and the substrate-derived C-8 atom and persists across most catalytic steps. While covalent catalysis is classically viewed as a means of substrate activation, the primary function of the transient covalent linkage in MoaC is to govern the spatial trajectory of the reacting carbon center. We term this catalytic strategy guiding covalent catalysis. This mechanism explains the long-standing absence of diffusible intermediates during MoaC catalysis and revises prevailing noncovalent models of Moco and pterin biosynthesis. Together, our findings establish guiding covalent catalysis as a distinct functional mode that enables precise spatial control in complex biochemical transformations and suggest that analogous guiding roles may operate in the biosynthesis of other cofactors.