Comprehensive In Silico Characterization of Imidazolonepropionase from Agrobacterium fabrum and Bacillus subtilis: Implications for Histidine Catabolism and Protein Stability

Abstract

Imidazolonepropionase (IPase), a pivotal enzyme in the histidine degradation pathway, catalyzes the hydrolysis of 4-imidazolone-5-propionic acid to N-formyl-L-glutamate. This comprehensive in silico investigation delves into the comparative characteristics of IPase from two phylogenetically distinct bacterial species: Agrobacterium fabrum (a Gram-negative bacterium, formerly Agrobacterium tumefaciens) and Bacillus subtilis (a Gram-positive bacterium). Utilizing an array of advanced bioinformatics and computational biophysics tools, including sequence analysis, physicochemical property prediction, structural comparisons, and molecular dynamics simulations, this study aims to unravel the subtle yet significant differences influencing their stability, compactness, and potential functional adaptations. Sequence analysis revealed a higher abundance of charged residues in B. subtilis IPase, contributing to increased polarity and hydrophilicity, which are often correlated with enhanced thermostability. While both enzymes exhibited a conserved βαβ fold and homodimeric architecture characteristic of the HutI superfamily, structural assessments indicated that B. subtilis IPase possessed a greater number of beta bulges, strands, and beta turns. Crucially, the B. subtilis enzyme demonstrated a propensity for forming more extensive network intra-protein interactions, including salt bridges and aromatic-aromatic interactions, compared to the predominantly isolated interactions found in A. fabrum IPase. Molecular dynamics simulations further substantiated these findings, showing that B. subtilis IPase exhibited lower root mean square deviation (RMSD) and root mean square fluctuation (RMSF) values, alongside a more compact radius of gyration (Rg) and a higher number of stabilizing hydrogen bonds. Conversely, A. fabrum IPase displayed higher solvent-accessible surface area (SASA) over extended periods, suggesting greater flexibility and potential for ligand interaction. These findings collectively indicate that IPase from Bacillus subtilis is inherently more stable and compact than its Agrobacterium fabrum counterpart, likely enabling more efficient histidine utilization, particularly under potentially adverse environmental conditions. This study not only deepens our understanding of IPase's structure-function relationship and evolutionary adaptations but also highlights the robust capabilities of in silico methodologies in guiding future experimental investigations and biotechnological applications related to microbial metabolism.