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Self-sufficient enzymes belong to the cytochrome P450 (CYP) group and are known for their superior hydroxylation catalytic activity. In the pursuit of identifying new pesticides to combat antimicrobial-resistant pathogens, we employed BM3 wild type (BM3-WT), the fastest monohydroxylating CYP, along with its seven homologs, to investigate the production of potential hydroxylated derivatives from the established pesticide, 4-oxocrotonic acid using high-pressure liquid chromatography (HPLC) method. Following the recombinant production of BM3-WT and three other homologs in E. coli, and their subsequent purification using Immobilized Metal Affinity Chromatography (IMAC), a novel enzyme assay approach was developed as a substitute for the carbon monoxide (CO) assay. This new method relied on the measurement of NADPH consumption at 340 nm by BM3-WT for palmitic acid. Leveraging this established technique, we explored the substrate specificity of BM3-WT and its homologs not only on palmitic acid but also on other structurally similar compounds, including 4-oxocrotonic acid. The results obtained from the established NADPH assay indicate that all tested enzymes displayed greater catalytic activity on 4-oxocrotonic acid in comparison to other substrates with similar structures. However, the impact of BM3-WT and its homologs on 4-oxocrotonic acid varied in terms of product specificity. Enzymes such as Poh, Trr and Bas-CYP D exhibited specificity in producing solely monohydroxylated products, while others tended to yield dehydroxylated and ketol metabolites.