Publications

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

  Khairy H., Wuebbeler J.H., Steinbuechel A.

  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 Nox_MI2 , which belongs to the old yellow ... >> More

  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 Nox_MI2 , which belongs to the old yellow enzyme (OYE) family, was identified. In the present study, it was proven that Nox_MI2 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 Nox_MI2 . Nox_MI2 showed a maximum specific activity (V_max ) of 3·36 μmol min^-1 mg^-1 corresponding to a k_cat of 2·5 s^-1 and an apparent substrate K_m of 0·6 mmol l^-1 , when different DTDB concentrations were applied. No metal cofactors were required. Moreover, Nox_MI2 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 Nox_MI2 revealed the presence of the cofactor FMN. Based on results obtained, Nox_MI2 adds a new physiological substrate and mode of action to OYE members.<h4>Significance and impact of the study</h4>It was unequivocally demonstrated in this study that an NADH:flavin oxidoreductase from Rhodococcus erythropolis MI2 (Nox_MI2 ) 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. Nox_MI2 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. << Less

  Lett. Appl. Microbiol. 63:434-441(2016) [PubMed] [EuropePMC]

• Biodegradation of the organic disulfide 4,4'-dithiodibutyric acid by Rhodococcus spp.

  Khairy H., Wubbeler J.H., Steinbuchel A.

  Four Rhodococcus spp. exhibited the ability to use 4,4'-dithiodibutyric acid (DTDB) as a sole carbon source for growth. The most important step for the production of a novel polythioester (PTE) using DTDB as a precursor substrate is the initial cleavage of DTDB. Thus, identification of the enzyme ... >> More

  Four Rhodococcus spp. exhibited the ability to use 4,4'-dithiodibutyric acid (DTDB) as a sole carbon source for growth. The most important step for the production of a novel polythioester (PTE) using DTDB as a precursor substrate is the initial cleavage of DTDB. Thus, identification of the enzyme responsible for this step was mandatory. Because Rhodococcus erythropolis strain MI2 serves as a model organism for elucidation of the biodegradation of DTDB, it was used to identify the genes encoding the enzymes involved in DTDB utilization. To identify these genes, transposon mutagenesis of R. erythropolis MI2 was carried out using transposon pTNR-TA. Among 3,261 mutants screened, 8 showed no growth with DTDB as the sole carbon source. In five mutants, the insertion locus was mapped either within a gene coding for a polysaccharide deacetyltransferase, a putative ATPase, or an acetyl coenzyme A transferase, 1 bp upstream of a gene coding for a putative methylase, or 176 bp downstream of a gene coding for a putative kinase. In another mutant, the insertion was localized between genes encoding a putative transcriptional regulator of the TetR family (noxR) and an NADH:flavin oxidoreductase (nox). Moreover, in two other mutants, the insertion loci were mapped within a gene encoding a hypothetical protein in the vicinity of noxR and nox. The interruption mutant generated, R. erythropolis MI2 noxΩtsr, was unable to grow with DTDB as the sole carbon source. Subsequently, nox was overexpressed and purified, and its activity with DTDB was measured. The specific enzyme activity of Nox amounted to 1.2 ± 0.15 U/mg. Therefore, we propose that Nox is responsible for the initial cleavage of DTDB into 2 molecules of 4-mercaptobutyric acid (4MB). << Less

  Appl. Environ. Microbiol. 81:8294-8306(2015) [PubMed] [EuropePMC]