In silico thermodynamics stability change analysis involved in BH₄ responsive mutations in phenylalanine hydroxylase: QM/MM and MD simulations analysis

Abstract

The mammalian tetrahydrobiopterin (BH₄)-dependent phenylalanine hydroxylases (PAH), involved in important metabolic pathways of phenylalanine, belong to non-heme iron-containing aromatic acid hydroxylases’ enzyme (AAH) family. AAHs utilize BH₄ as protein co-factor and thus promote hydroxylation reactions of their substrates. Any alterations in BH₄ -mediated AAH’s pathway or mutations in these enzymes are responsible for various disorders, and thus highlights the importance of mutational analysis to assess the effect on their biosynthetic pathways. Our present studies are aimed at single-site mutations in PAH that lead to thermodynamic stability change upon folding and further validation of designed non-reduced BH₂ designed co-factors. We have presented single-site mutational analysis of PAH where single-site mutations have been identified from known literature. Further, in silico studies with the PAH, in silico mutant PAH, and crystallized known mutant A313T forms, involved QM/MM and Molecular Dynamics (MD) simulations analysis. The modified co-factor A showed high affinity with PAH and all mutant PAH with high G-score of −14.851. The best pose high affinity co-factor A subjected to QM/MM optimization which leads to square-pyramidal coordination of non-heme active site. The structural and energetic information obtained from the production phase of 20 ns MD simulation of co-factor-metalloprotein complex results helped to understand the binding mode and involvement of three molecules throughout the reaction pathways’ catalysis of PAH. The free energies of binding (dG) of A were found to be −68.181 kcal/mol and −72.249 for 1DMW and 1TDW for A313T mutant. Binding of Co-factor A do not perturb the coordination environment of iron at the active site which resides in 2-Histdine and 1-Glutamate triad, and may enhance the percentage response towards co-factor-mediated therapy.

Keywords:

• docking
• hydroxylases
• mutation
• QM/MM
• MD
• mutation
• tetrahydrobiopterin

Acknowledgments

We are grateful to Dr R P Tripathi, Director, Institute of Nuclear Medicine and Allied Sciences (INMAS), Delhi for providing the necessary facilities for these studies under the project INM. 311 (3.1) and Council of Scientific and Industrial Research University of Grant commission (CSIR-UGC) for providing financial assistance to the first author.