Molecular Basis for Cγ-N Bond Formation by PLP-Dependent Enzyme LolC

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