AKR1C3-driven restoration of tetrahydrobiopterin synthesis in cellular sepiapterin reductase deficiency

Sepiapterin reductase (SPR) catalyzes several key steps in the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthases and aromatic amino acid hydroxylases, and therefore for neurotransmitter production. Although several reductases-including carbonyl reductase 1 (CBR1), aldose reductase (AKR1B1), and AKR1C3-can substitute for SPR activity in vitro, their physiological significance remains unresolved. This study examines AKR1C3 as a component of an alternative BH4-generating pathway and evaluates its capacity to compensate for BH4 loss under diminished SPR activity. In vitro assays identified 2'-OXPH4 as the primary product of AKR1C3, redirecting pathway flux away from the canonical 1'-OXPH4 intermediate and the sepiapterin-salvage pathway. To assess the occurrence and efficiency of this route in cells, we generated SPR-knockout (SPR-KO) cell and evaluated pathway products in SPR-KO and wild-type (WT) backgrounds. In WT cells neither AKR1C3 nor SPR overexpression altered BH4 synthesis, indicating that neither enzyme is rate-limiting. In contrast, AKR1C3 increased BH4 levels in SPR-KO cells, while inhibiting sepiapterin production, revealing that AKR1C3 becomes functionally engaged only when SPR activity is decreased. Based on relative enzyme abundance, AKR1C3 and SPR exhibited comparable catalytic efficiency in this context. Importantly, AKR1C3-mediated BH4 production was sufficient to sustain tyrosine hydroxylase (TH) activity in SPR-KO cells, as demonstrated by L-DOPA formation. These findings establish AKR1C3-driven 2'-OXPH4 synthesis as a bona fide, inducible pathway capable of maintaining BH4 levels when SPR activity is limiting. This alternative path provides a compelling therapeutic target and introduces a new diagnostic consideration for patients with diminished SPR activity.