Catalytic Mechanism of γ-Elimination and Michael Addition Catalyzed by Pyridoxal-5'-Phosphate-Dependent γ-Substitution Enzyme Fub7 Involved in the Biosynthesis of 5-Alkyl-pipecolic Acid.

Fub7 is a rare pyridoxal 5'-phosphate (PLP)-dependent enzyme that can subsequently catalyze the γ-elimination of O-acetyl-l-homoserine (OAH) and Michael addition with n-valeraldehyde (NVA) to synthesize 5-alkyl-pipecolic acid, which shows significant potential in synthetic chemistry. In this study, we constructed the computational models and performed MD and QM/MM calculations to illuminate the complete catalytic cycle of Fub7, which contains five reaction stages, including external aldimine formation, γ-elimination, Michael addition, reformation of the internal aldimine, and final cyclization and dehydration, of which γ-elimination and Michael addition correspond to relatively high energy barriers. In addition, according to our test calculations, Fub7 can also catalyze the β-elimination. During the catalysis, the terminal amino of Lys211 exhibits significant conformational changes and was found to play multiple roles. On one hand, Lys211 forms internal aldimine with the cofactor PLP. On the other hand, Lys211 functions as a catalytic base/acid or as a bridge to mediate a series of proton transfer in the γ-elimination and Michael addition. Tyr109 only participates in the Michael addition. The deprotonated Tyr109 acts as a base to abstract the α-proton of NVA to generate the carbanion nucleophile, which is the key for Michael addition. These findings may provide useful information for understanding the catalysis of PLP-dependent enzymes and designing biocatalysts for γ-substitution.