Structural insights into the hydrolysis and polymorphism of methotrexate polyglutamate by zebrafish γ-Glutamyl hydrolase

γ-Glutamyl hydrolases (γGH) catalyze the hydrolysis of γ-linked glutamate residues from the polyglutamyl of folates and antifolates, such as methotrexate (MTX), a widely used anticancer drug. We describe the first crystal structures of the endopeptidase-type γGH (zγGH) from zebrafish and the mutant complexes with MTX(Glu)₅ and hydrolyzed MTX(Glu)₁, revealing the complete set of key residues involved in hydrolysis as well as the substrate-binding subsites (-1 to +2). The side chain of Phe20 and the 6-methylpterin ring of MTX(Glu)₅ invoke π-π interactions to promote distinct concerted conformational alterations involving ∼90 rotations in the complexes with the zγGH-C108A and zγGH-H218N mutant proteins. The structural geometries of the MTX(Glu) ₅ and hydrolyzed MTX(Glu)₁ in the mutant complexes differ significantly from those of the previously known MTX(Glu)₁, providing polymorphic information. Together with the structural comparison and the activity analysis, these results shed light on the catalytic mechanism and substrate recognition of zγGH and other γ-glutamyl hydrolases.