The NADH:flavin oxidoreductase Nox from Rhodococcus erythropolis MI2 is the key enzyme of 4,4'-dithiodibutyric acid degradation.

The reduction of the disulphide bond is the initial catabolic step of the microbial degradation of the organic disulphide 4,4'-dithiodibutyric acid (DTDB). Previously, an NADH:flavin oxidoreductase from Rhodococcus erythropolis MI2 designated as NoxMI2 , which belongs to the old yellow enzyme (OYE) family, was identified. In the present study, it was proven that NoxMI2 has the ability to cleave the sulphur-sulphur bond in DTDB. In silico analysis revealed high sequence similarities to proteins of the flavin mononucleotide (FMN) reductase family identified in many strains of R. erythropolis. Therefore, nox was heterologously expressed in the pET23a(+) expression system using Escherichia coli strain BL21(DE3) pLysS, which effectively produces soluble active NoxMI2 . NoxMI2 showed a maximum specific activity (Vmax ) of 3·36 µmol min-1  mg-1 corresponding to a kcat of 2·5 s-1 and an apparent substrate Km of 0·6 mmol l-1 , when different DTDB concentrations were applied. No metal cofactors were required. Moreover, NoxMI2 had very low activity with other sulphur-containing compounds like 3,3'-dithiodipropionic acid (8·0%), 3,3'-thiodipropionic acid (7·6%) and 5,5'-dithiobis(2-nitrobenzoic acid) (8·0%). The UV/VIS spectrum of NoxMI2 revealed the presence of the cofactor FMN. Based on results obtained, NoxMI2 adds a new physiological substrate and mode of action to OYE members. SIGNIFICANCE AND IMPACT OF THE STUDY: It was unequivocally demonstrated in this study that an NADH:flavin oxidoreductase from Rhodococcus erythropolis MI2 (NoxMI2 ) is able to cleave the xenobiotic disulphide 4,4'-dithiodibutyric acid (DTDB) into two molecules of 4-mercaptobutyric acid (4MB) with concomitant consumption of NADH. NoxMI2 showed a high substrate specificity as well as high heat stability. This study provides the first detailed characterization of the initial cleavage of DTDB, which is considered as a promising polythioester precursor.