Methionine γ-lyase traps cofactor pyridoxal-5'-phosphate through specific serine-mediated affinity

Pyridoxal-5'-phosphate (PLP), the ubiquitous and ancient cofactor, plays important roles in enzymatic elimination, transamination and other reactions. The catalytic efficiency of PLP-dependent enzymes is significantly higher than that of free PLP. The recruitment of solution PLP by the enzymes, particularly through interactions outside the active-site entrance, is the key step determining the occurrence of PLP-mediated catalysis. However, the precise mechanism by which enzymes recruit solution PLP remains elusive.

This study aims to elucidate the mechanism by which enzymes, specifically methionine γ-lyase (MGL) that suppresses cancer cell proliferation through serum or dietary methionine depletion, recruit solution PLP and to investigate the role of the C-terminal domain in this process. The study also seeks to explore the relationship between PLP recruitment and the efficient L-methionine catabolism in the host organism.

We report the crystal structure of yMGL, which belongs to a newly identified subgroup of cystathionine γ-lyases, in complex with L-methionine and PLP. To investigate the functional role of the C-terminal domain in PLP recruitment and enzyme activity, we performed C-terminal domain truncations and mutations and assessed their effects both in vitro and in vivo, analyzing changes in PLP binding affinity and L-methionine catabolism, respectively.

Through structural, biochemical, bioinformatic and metabolic analyses, we demonstrate that the C-terminal domain of yMGL, outside the canonical PLP-binding domain, is essential for the specific interaction between yMGL and PLP, and L-methionine catabolism. A conserved Ser residue within this domain, located outside of active-site entrance, determines PLP recruitment.

These findings elucidate a previously uncharacterized mechanism of PLP recruitment by MGLs and enable rational design of MGLs with enhanced PLP binding and catalytic performance.