Structural and Mechanistic Insights into Fgm3-Catalyzed Cβ−Cγ Bond Cleavage of an Amino Acid

Pyridoxal 5′-phosphate (PLP)-dependent enzymes represent one of the most functionally diverse families of biocatalysts, mediating a variety of bond transformations associated with amino acids. However, catalytic strategies that enable C−C bond cleavage at remote positions within amino acids remain exceedingly rare. Recently, we identified Fgm3, an unusual PLP-dependent enzyme in Fusarium graminearum, which catalyzes the Cβ−Cγ bond cleavage of 4(S)-hydroxy-l-arginine, yielding l-alanine and guanidino acetaldehyde during the biosynthesis of the fungal virulence factor fusaoctaxin B. Here, using X-ray crystallography, site-specific mutagenesis, and theoretical computation, we provide evidence that rationalizes the retro-aldol-like reaction catalyzed by Fgm3. We determined six high-resolution crystal structures of Fgm3, in complex with PLP and either its substrate, a substrate analog, or its product. These structures reveal a preorganized substrate-binding pocket that can anchor the α-carboxylate and guanidino for precise recognition. Combined structural and mutational analyses identified residue His116 as the key catalytic base responsible for γ-hydroxyl deprotonation to trigger Cβ−Cγ bond cleavage. Theoretical computation delineates the catalytic energy landscape and accordingly supports a synergistic catalytic mechanism in which His116 orchestrates proton transfer and C−C bond breaking. These findings establish the molecular basis of Fgm3-mediated C−C cleavage, expand the repertoire of PLP enzymology, and provide a blueprint for engineering biocatalysts to access noncanonical amino acids.