Computational Study of Pathogenic Variants in Phenylalanine-4-hydroxylase (PAH): Insights into Structure, Dynamics, and BH Binding.

The phenylalanine-4-hydroxylase (PAH) gene encodes the PAH enzyme, which is necessary for the conversion of l-phenylalanine (l-Phe) to l-tyrosine (l-Tyr). Deleterious mutations in PAH can disrupt its function, leading to the toxic buildup of phenylalanine in the brain and causing the inherited genetic disorder phenylketonuria (PKU). This condition results in behavioral issues, epilepsy, and intellectual disability. This in silico study has been conducted to investigate the structure and dynamics of both wild-type PAH and its variants, including I65T and R408W, which are prevalent in U.S. patients, as well as D282G and A202T, commonly observed in PKU patients from China and Korea. SIFT, PolyPhen-2, PhD-SNP, and MutPred2 methods, which utilize either sequence-based or machine learning algorithms, predicted the four mutations to be disease-causing and deleterious with the potential to disrupt the PAH structure and impair its function, thereby confirming their association with PKU. Four replicates of 500 ns molecular dynamics (MD) simulations resulting in a cumulative simulation time of 2 μs for all variants demonstrated that all these variants adversely affect the PAH structure and dynamics. The MM/GBSA binding free energy of I65T with BH, a crucial cofactor in the hydroxylation of l-Phe to l-Tyr, is found to be -12.8 kcal/mol compared to -16.5 kcal/mol for the wild type. Similarly, the R408W variant decreased BH binding with a calculated free energy of -11.4 kcal/mol. Additionally, the binding affinity of the tetramerization domains in R408W significantly reduced by at least 26.7 kcal/mol compared to the wild type. This study highlights how different pathogenic mutations in PAH impact the protein's structure, dynamics, and binding affinity, possibly leading to advancements in targeted drug development for PKU.