Identification of a key amino acid residue in the cofactor-independent oxidase ActVA-ORF3 involved in actinorhodin biosynthesis using AlphaFold2-predicted modeling and mutational analysis

Actinorhodin (ACT) is a polyketide antibiotic with a dimerized pyronaphthoquinone (PNQ) skeleton, produced by Streptomyces coelicolor A3(2), a preferred model organism for in-depth studies on antibiotic biosynthesis and its regulation in actinomycetes. The cofactor-independent oxidase ActVA-ORF3 participates in the dimerization step of ACT biosynthesis and oxidizes both monomeric and dimeric substrates. We conducted functional analysis of ActVA-ORF3 using AlphaFold2-based structural modeling, followed by mutational analysis. Glutamate 164 (E164) was identified as a key residue, likely functioning as a catalytic base. Docking simulation (DS) suggested that both monomer and dimer substrates are recognized as naphthalene-type isomers and that E164 interacts with the hydroxyl groups of both substrates. Further DS of a molecular oxygen with the substrate-enzyme complex provides a reasonable model that can explain the hydride transfer from the substrate leading to a hydride addition to the oxygen molecules, followed by protonation and the formation of hydrogen peroxide. Additionally, genomic database analysis revealed the distribution of gene clusters containing homologs of both act VA-ORF3 and type II polyketide synthase genes, suggesting the existence of polyketide-based secondary metabolic pathways involving an oxidative mechanism similar to that of ActVA-ORF3. • AlphaFold2 modeling identified a key catalytic residue in ActVA-ORF3. • E164 is critical for oxidative dimerization in actinorhodin biosynthesis. • Docking simulation revealed substrate interaction and reaction mechanism. • Mutational analysis confirmed the catalytic role of E164 in ActVA-ORF3. • Gene cluster analysis suggests conserved oxidative steps in polyketide pathways.