In the last few years, much attention has been drawn to emerging contaminants due to their severe effects on human health and the lack of information about them. Among them, erythromycin has risen as a potential threat in developing antimicrobial resistance, and being capable of detecting it in water bodies can lead to the generation of measurements capable of degrading it.

In our project, erythromycin C-12 hydroxylase is used as a detector for the presence of erythromycin by catalyzing the oxidation of two stereoisomers of erythromycin, erythromycin B and D, to erythromycin C, requiring NADPH as a reagent and, therefore, obtaining NADP+ as a reaction product. Consequently, it is possible to evaluate the presence of erythromycin through a coupled reaction employing a NADP+/NADPH colorimetric assay. The following image shows the complete reaction according to Stassi and collaborators (1993):

Erythromycin C-12 hydroxylase is a monomer with 397 amino acids in length. According to Savino et al. (2009), it binds one heme b(iron(II)-protoporphyrin IX) group per subunit as a cofactor. Lambalot et al. (1995) reported a Michaelis constant of 8 μM for erythromycin D, concluding that it shows a 1200-1900-fold preference for erythromycin D over the alternative substrate erythromycin B. This enzyme participates in various molecular and biological processes, ranging from macrolide biosynthetic processes to oxidoreductase reactions. Next, we present the three-dimensional structure of mVenus generated by AlphaFold2 using MMSeqs2 (Mirdita et al., 2022). This structure is as follows: