Azo dyes constitute major pollutants in textile dyeing wastewater, where cleavage of the azo bond (-N = N-) represents the critical step in their degradation [1]. Microbial azoreductases have emerged as pivotal biocatalysts in bioremediation technologies due to their high efficiency in catalyzing azo bond reduction [2]. These enzymes are widely distributed among aerobic and anaerobic bacteria, functioning through a coenzyme (NADH/NADPH)- and flavin cofactor (FMN/FAD)-dependent electron transfer chain: NAD(P)H donates electrons, which are relayed via the flavin isoalloxazine ring to the dye molecule, ultimately cleaving the chromophore [3]. Based on flavin dependency and coenzyme specificity, azoreductases are classified into three types: Type I: FMN-dependent homodimers preferentially utilizing NAD; Type II: FMN-dependent enzymes requiring NADPH as the electron donor; Type III: FMN-independent monomeric enzymes exclusively utilizing NADPH [4].

Flavoproteins serve as electron "shuttles" through the redox properties of their isoalloxazine rings. FMN, owing to its smaller molecular size and conformational flexibility, exhibits superior compatibility with substrate-binding pockets [5]. Studies indicate that conserved aromatic residues within the flavin domain are essential for maintaining isoalloxazine ring conformation—they stabilize the flavin ring plane through π-π stacking and optimize electron transfer pathways [6]. Despite the resolution of the first azoreductase crystal structure (EcAzoR) in 2006, structural information remains unavailable for over 80 % of annotated azoreductases, obscuring their catalytic mechanisms and substrate specificity determinants [7]. Recent advances in metaproteomics have accelerated novel enzyme discovery, while AI-based structure prediction (e.g., AlphaFold2) offers new avenues for resolving protein tertiary structures. Integration of bioinformatics tools (e.g., conserved residue analysis, molecular docking) enables in silico screening of potential functional sites to guide protein engineering [8], [9].

The target enzyme of this study, BVU5, originates from the decolorizing microbial consortium DDMZ1 [10]. Its primary sequence (UniParc: UPI0002297456) is designated GbcB in NCBI. Previous work confirmed BVU5 as an FMN-dependent Type II azoreductase with broad-spectrum decolorization capability. Its UniParc accession and NCBI designation classify it within the flavin reductase superfamily, yet its structure-function relationships remain uncharacterized. Notably, the flavin domain of BVU5 harbors a highly conserved Tyr69 residue. Whether Tyr69 modulates electron transfer efficiency by stabilizing FMN conformation—thereby governing decolorization performance—warrants in-depth investigation. To address this, we employed a "computational prediction, experimental validation, mechanistic elucidation" strategy: Constructed a high-confidence BVU5 model using AlphaFold2 and defined its conserved locus; Generated site-directed mutants (Y69F,Y69C) and quantified decolorization efficiency against 12 dyes (azo/anthraquinone/triphenylmethane types); Elucidated FMN-binding pattern alterations and electron transfer routes via molecular docking and interaction analysis. This study provides mechanistic insights into how conserved flavin domain residues regulate electron transfer and establishes a foundation for designing thermostable, broad-spectrum decolorizing enzymes.