Dimerization of MilM Is Essential for Catalyzing the Pyridoxal-5'-phosphate (PLP)-Dependent Cγ-Hydroxylation of...

MilM from the mildiomycin biosynthetic pathway is a PLP-dependent enzyme, previously annotated as an aminotransferase, but has recently been demonstrated as l-arginine oxidase cum C-hydroxylase. Here, we report detailed biochemical, biophysical, structural modeling, and molecular dynamics (MD) simulation-based investigations of MilM from Streptoverticillium rimofaciens B-98891 to elucidate the mechanisms of substrate binding and catalysis and the roles of the active-site residues involved in these processes. Our experimental results confirmed that MilM functions as a stable homodimer, requiring the PLP cofactor and molecular oxygen to transform the l-arginine substrate into 5-guanidino-4-hydroxy-2-oxovaleric acid and 5-guanidino-2-oxovaleric acid via a possible superoxide radical anion intermediate, while generating H₂O₂ and NH₃ as reaction byproducts. Our labeling studies also established that the hydroxyl group in the product is obtained from the solvent, water. Structural modeling, MD simulation, and site-directed mutagenesis reveal that residues from both protomers contribute to cofactor and substrate stabilization, including Lys232 and His31 as key catalytic residues as well as additional residues forming an extended active-site interaction network. Moreover, MD simulations uncovered a dimer-mediated alternating-lid mechanism in which coordinated motions at the dimer interface regulate substrate access and product release through transient tunnels while maintaining a protected catalytic environment. This seesaw-like dynamics provides a mechanistic framework for understanding how dimerization controls enzyme function, a critical phenomenon previously left unnoticed in similar PLP-dependent oxidases/hydroxylases. Overall, these findings provide new insights into substrate/cofactor stabilization and the catalytic mechanism of MilM and expand our understanding of the remarkable PLP-dependent oxidases and hydroxylases in natural product biosynthesis.