Molecular Basis\nfor Cγ‑N Bond Formation\nby PLP-Dependent Enzyme LolC

Pyridoxal 5′-phosphate (PLP)-dependent enzymes\ncatalyze\na diverse array of biochemical transformations, making them invaluable\nbiocatalytic tools for the synthesis of complex bioactive compounds.\nHere, we report the biochemical characterization of LolC, a PLP-dependent\nγ-synthase involved in the biosynthesis of loline alkaloids.\nLolC catalyzes the formation of a Cγ-N bond between <i>O</i>-acetyl--homoserine (OAH) and l-proline, generating\na diamino diacid intermediate. Our findings reveal that LolC exhibits\nstrict specificity for proline and its analogues, contrasting with\nthe substrate promiscuity of closely related Cγ-C bond-forming\nenzyme Fub7. Structural analysis, using an AlphaFold model, identifies\nkey differences in the substrate entrance tunnel of LolC, which is\namphiphilic and distinct from the hydrophobic tunnel in Fub7. A mutagenesis\nstudy further highlights the functional divergence of a key active\nsite residue between these enzymes. These results provide new insights\ninto the substrate specificity and catalytic function of LolC, offering\na valuable comparison to Fub7 and advancing our understanding of PLP-dependent\nenzymes involved in γ-substitution reactions.