Comparative Sequence and Structural Analysis of ACMSD-ACMS Binding in Kynurenine Pathway

Abstract

The Kynurenine pathway is a crucial metabolic pathway, which plays a pivotal role in regulating various physiological processes, including immune response and neurotransmitter synthesis. During this pathway, an enzyme ACMSD (α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase) plays a crucial role by catalyzing the conversion of α-amino-β-carboxymuconate ε-semialdehyde (ACMS) into quinolinic acid and regulates the balance of neuroactive metabolites. Despite its central role, the structural and functional aspects of ACMSD remain largely unexplored. In this study, we employed comparative modeling, molecular docking, and molecular dynamics simulations to unravel the intricate details of ACMSD interactions and their implications across species. Amino acid sequences of ACMSD from various organisms were obtained through UniProtKB. X ray structure of human ACMSD was retrieved from the Protein Data Bank (PDB). Multiple Sequence Alignment (MSA) was performed using ClustalW and Clustal Omega to identify the conserved residues in ACMSD. Homology modeling was performed to predict ACMSD structures for wolf, cheetah, dog, rabbit and mouse using MODELLER and SWISS-MODEL. UCSF Chimera was used for energy minimization of all the predicted protein structures. WinCoot and MolProbity tools were employed to validate and refine the protein structures. Intra-Protein interactions in ACMSD dimer were scrutinized, uncovering a network of vital hydrophobic interactions and hydrogen bonding patterns between the constituent chains. The evaluation of ACMS as a substrate for ACMSD involved assessing its physicochemical properties using Lipinski's Rule of Five. These results demonstrated that ACMS adhered to drug-like characteristics, with its molecular weight, hydrogen bond donors/acceptors, LogP, and PSA lie within acceptable ranges. ACMS structure was obtained using PubChem. Avogadro and Open Babel tools were used for energy minimization. Ligand-protein docking was performed using AutoDock Vina, HDock, and PatchDock. LigPlot+ and Discovery Studio were used for visualization and interaction analysis. GROMACS was utilized for MD simulations to explore the dynamic behavior of docked complexes. Preparation of PDB files, topology file generation, PBC, solvation, addition of ions, energy minimization, equilibration, and MD simulation runs were performed. Root mean square deviation and root mean square fluctuation calculations were performed to assess stability and fluctuations during MD simulations. Comparative Sequence and Structural Analysis of ACMSD-ACMS Binding in Kynurenine Pathway viii Abstract The structural conservation observed among ACMSD proteins across species underscores their significance in maintaining the integrity of the Kynurenine pathway. The conservation of binding residues implies a common mechanism of substrate recognition. Specific residues, including ARG47, TRP188, TRP191, ASP291, PHE294, and PRO295 in one subunit, as well as ARG235 and ARG243 in the other subunit, consistently play crucial roles in the binding process. The dynamic behavior observed through simulations offers insights into the dynamic interplay between ACMSD and ACMS. The relatively low RMSD values during the simulation periods indicated stable system during simulation. Fluctuations in specific residues were observed, particularly in the loop regions, while binding residues remained relatively stable. These fluctuations might be indicative of inherent flexibility required for the efficient substrate binding. The conformational changes observed in both hACMSD and wACMSD during MD simulations suggest potential structural adaptations that may accommodate substrate binding. In conclusion, this study provides a comprehensive analysis of ACMSD-ACMS interactions among multiple organisms, thereby shedding light on the Kynurenine pathway. The results offer a foundation for future experimental validation, potentially leading to therapeutic interventions that may target this critical pathway. By unveiling the structural and dynamic aspects of ACMSD, this study contributes to our understanding of metabolic regulation and opens new avenues for further studies