A Systematic In-Silico Screening of Bacterial Αlpha Galactosidases: Integrating Structural, Functional, and Immunoinformatic Analyses to Identify Potential Therapeutic Candidates

This study aims to identify novel enzyme candidates from bacterial sources to enhance the therapeutic potential of human alpha-galactosidase A (α-Gal). The limitations of current enzyme replacement therapies for Fabry disease, such as immunogenicity, necessitate the search for alternative homologs with superior properties. In this context, a BLASTp search using human α-Gal as a reference identified 100 potential bacterial homologs. The three-dimensional structural models of these homologs were subjected to a rigorous quality control process using the SAVES server, and candidates with inadequate structural integrity were eliminated. The immunogenic potential of the selected candidates was assessed by predicting B-cell epitopes via the ElliProt server. For functional analysis, molecular docking simulations were performed with the natural substrate, globotriaosylceramide (Gb3), and the artificial substrate, p-nitrophenyl α-galactopyranoside (pNP-Gal). The results highlighted proteins such as A0A1M5FVV3 and Q5LFG6, which showed the highest binding affinity for the Gb3 substrate, and proteins like R6DB23 and R5RG66, which exhibited the highest affinity for the pNP-Gal substrate. Furthermore, the interactions of conserved Aspartate residues, which play a key role in substrate binding and are critical for catalytic activity, were confirmed. This study identifies specific bacterial α-Gal homologs that combine high substrate affinity with low immunogenicity potential as promising candidates for further experimental validation as next-generation biotechnological and novel bacterial homologs for Fabry disease.