Integrated molecular simulations reveal channel-guided PLP loading and prereactive preorganization in human ornithine decarboxylase

Ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine biosynthesis, requires pyridoxal 5’-phosphate (PLP) binding and internal aldimine formation prior tocatalysis. Although the chemical steps of PLP-dependent activation have been widely investigated, the physical process by which PLP is captured from the protein exterior, delivered into the buried catalytic cavity, and preorganized for reaction remains poorly understood. Here, we employed an integrated molecular simulation workflow to elucidate the pathway, energetics, and residue-level determinants of PLP delivery in human ODC. The results show that PLP loading is not a passive diffusion event, but a channel-guided and energetically biased activation process governed by cooperative electrostatic steering, hydrogen-bond relay, and aromatic orientation control. A dominant delivery route with the lowest mechanical resistance was identified, along which PLP undergoes sequential recognition, inward migration, conformational reorientation, and final accommodation. This staged process positions PLP in a prereactive state suitable for internal aldimine formation, thereby linking cofactor transport directly to catalytic readiness. Mechanistically, these findings expand the conventional view of ODC activation from a reaction-centered process to a coupled delivery-preorganization mechanism. More broadly, the principles revealed here may help explain how PLP-dependent enzymes with buried active sites achieve selective cofactor loading and may provide new conceptual opportunities for modulating enzyme activity by perturbing cofactor delivery rather than only targeting substrate binding or covalent chemistry.