title: The NADH:flavin oxidoreductase Nox from Rhodococcus erythropolis MI2 is the key enzyme of 4,4'-dithiodibutyric acid degradation.
abstract: 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 NoxMI2has 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. NoxMI2showed a maximum specific activity (Vmax) of 3·36 μmol min-1mg-1corresponding to a kcatof 2·5 s-1and an apparent substrate Kmof 0·6 mmol l-1, when different DTDB concentrations were applied. No metal cofactors were required. Moreover, NoxMI2had 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 NoxMI2revealed the presence of the cofactor FMN. Based on results obtained, NoxMI2adds a new physiological substrate and mode of action to OYE members.
  
  
  CONCLUSIONS
  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. NoxMI2showed 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.
authors: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Münster, Germany.|Botany and Microbiology Department, Faculty of Science, Alexandria University, Alexandria, Egypt.
authors: H
authors: H
authors: Khairy
authors: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Münster, Germany.
authors: J H
authors: JH
authors: Wübbeler
authors: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Münster, Germany.|Faculty of Environmental Sciences, King Abdulaziz University, Jeddah, Saudi Arabia.
authors: A
authors: A
authors: Steinbüchel
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001673:Biodegradation, Environmental; D020148:Butyric Acid / Q000737:chemistry / Q000378:metabolism*; D038181:FMN Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005415:Flavins / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D019255:NADPH Oxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism; D011422:Propionates / Q000378:metabolism; D012240:Rhodococcus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013455:Sulfur / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005415:Flavins; D011422:Propionates; D009243:NAD; D020148:Butyric Acid; C013298:thiodipropionic acid; D013455:Sulfur; D010088:Oxidoreductases; D038181:FMN Reductase; D019255:NADPH Oxidases
keywords: 
  Rhodococcus erythropolis
  ; 4,4′-dithiodibutyric acid; 4-mercaptobutyric acid; disulphide reductase; old yellow enzyme family; polythioester