DNAJC12 Stabilizes Phenylalanine Hydroxylase and Facilitates Its Substrate‐Dependent Activation

ABSTRACT Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin (BH 4 )‐dependent enzyme that converts L‐phenylalanine (L‐Phe) to L‐tyrosine. PAH dysfunction leads to the accumulation of L‐Phe in the blood (hyperphenylalaninemia; HPA), which may reach neurotoxic levels, resulting in phenylketonuria (PKU). PKU is associated with pathogenic variants in PAH, mostly causing misfolding and instability, leading to decreased levels of PAH protein and activity. Recently, variants in the J‐domain protein DNAJC12 have also been associated with HPA in patients, demonstrating the importance of protein homeostasis regulation for proper PAH function. DNAJC12 and PAH have previously been reported to interact, but the molecular and structural mechanisms behind complex formation have remained unclear. In this work, we show that DNAJC12 binds to PAH but presents higher affinity for its L‐Phe activated form, which resembles the conformation of unliganded tyrosine hydroxylase, a structurally and functionally related enzyme that also binds to DNAJC12. At saturation, four DNAJC12 monomers bind and stabilize the PAH tetramer, protecting it from aggregation and lowering the L‐Phe concentration necessary for substrate‐induced activation, without affecting the interaction of the enzyme with its cofactor BH 4 . Importantly, DNAJC12 also stabilizes and delays the aggregation of the PKU‐associated variant PAH‐p.R261Q. This study provides the first detailed characterization of the molecular determinants driving PAH:DNAJC12 complex formation and reveals how this interaction modulates enzyme stability and activity, and stimulates Hsc70 ATPase activity. These findings provide mechanistic insight into the pathogenic basis of DNAJC12 deficiency and identify the PAH:DNAJC12 complex as a promising therapeutic target for HPA.