Comparative Structural Modeling Suggests Distinct Signatures of Conformational Plasticity and Surface Physicochemistry in Phytoene Synthase and Dehydrosqualene Synthase.

Carotenoids are essential metabolites involved in photosynthesis, cellular protection, pigmentation, and antioxidant activities. Phytoene synthase (PSY/CrtB) utilizes C20 substrates in carotenoid biosynthesis, whereas its structural homolog, dehydrosqualene synthase (CrtM), preferentially accepts C15 substrates. Although previous studies have identified CrtM mutations that expand substrate scope, the molecular basis of substrate discrimination in PSY/CrtB remains poorly understood, largely because of the absence of experimentally determined three-dimensional structures. Here, we integrated comparative sequence analysis, homology modeling, and molecular dynamics (MD) simulations to investigate the structural basis of substrate discrimination in PSY/CrtB. Comparative sequence analysis suggested distinct overall conservation landscapes in PSY/CrtB and CrtM, with 20 highly conserved positions shared between them and clustered around the catalytic core. MD simulations suggest that PSY models exhibit minimal differentiation under cross-ligand conditions, consistent with its greater conformational plasticity. Surface property analysis suggested hydrophobic patches and an amphipathic helix (Helix-13) in PSY that were preferentially conserved in PSY homologs relative to CrtM homologs. Taken together, our analyses suggest that greater conformational plasticity may facilitate the accommodation of C20 substrates in PSY and that its conserved hydrophobic surface architecture may shape its surface physicochemistry. These findings suggest that differences in substrate accommodation between PSY/CrtB and CrtM may reflect coordinated variation in conformational dynamics, pocket hydrophobicity, and surface architecture, rather than substantial alterations to the conserved catalytic core.