A dibenzothiophene (DBT)-degrading bacterium, Rhodococcus erythropolis D-1, which utilized DBT as a sole source of sulfur, was isolated from soil. DBT was metabolized to 2-hydroxybiphenyl (2-HBP) by the strain, and 2-HBP was almost stoichiometrically accumulated as the dead-end metabolite of DBT degradation. DBT degradation by this strain was shown to proceed as DBT-* DBT sulfone-2-HBP. DBT at an initial concentration of 0.125 mM was completely degraded within 2 days of cultivation. DBT at up to 2.2 mM was rapidly degraded by resting cells within only 150 min. It was thought this strain had a higher DBTdesulfurizing ability than other microorganisms reported previously.
The release of SO2 from petroleum products derived from crude oil, which contains sulfur compounds such as dibenzothiophene (DBT), leads to air pollution. The ‘4S’ metabolic pathway catalyzes the sequential conversion of DBT to 2‐hydroxybiphenyl via three enzymes encoded by the dsz operon in several bacterial species. DszC (DBT monooxygenase), from Rhodococcus erythropolis D‐1 is involved in the first two steps of the ‘4S’ pathway. Here, we determined the first crystal structure of FMN‐bound DszC, and found that two distinct conformations occur in the loop region (residues 131–142) adjacent to the active site. On the basis of the DszC–FMN structure and the previously reported apo structures of DszC homologs, the binding site for DBT and DBT sulfoxide is proposed. Database The atomic coordinates and structure factors for apo‐DszC (PDB code: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=3X0X) and DszC‐FMN (PDB code: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=3X0Y) have been deposited in the Protein Data Bank (http://www.rcsb.org).
The occurrence of S‐adenosylhomocysteine hydrolase (EC 3.3.1.1) was found in a variety of prokaryotes. These prokaryotes did not exhibit any activities of S‐adenosylhomocysteine nucleosidase (EC 3.2.2.9) and S‐ribosyl‐homocysteine hydrolase (EC 3.3.1.3), which had been the generally accepted prokaryote enzymes for the regeneration of free homocysteine from S‐adenosylhomocysteine in the activated methyl cycle. In these prokaryotes S‐adenosylhomocysteine hydrolase was suggested to be the only enzyme functioning for the regeneration of free homocysteine by enzymological and immunochemical studies. S‐Adenosylhomocysteine hydrolase was purified and crystallized from cells of a prokaryote, Alcaligenes faecalis. The purified enzyme was found to be homogenous on ultracentrifugation and gel electrophoresis. Its relative molecular mass is approximately 280000 and it is composed of six identical subunits with a Mr of approximately 48000. The NH2‐terminal and COOH‐terminal amino acids are lysine and tyrosine respectively. The enzyme contains 6 ma1 NAD/mol. Some nucleosides, such as formycin A, nebularine, adenosine N1 oxide and so on, are able to substitute for adenosine yielding the corresponding S‐nucleosidylhomocysteine congeners. Modification of the 5′‐hydroxymethyl group in adenosine leads to the most potent inhibition of the thioether formation of homocysteine with adenosine. The enzyme from A. faecalis has some immunological similarities to other prokaryote S‐adenosylhomocysteine hydrolases, but is different from the enzymes of animal sources.
The dibenzothiophene (DBT)-desulfurizing bacterium, Rhodococcus erythropolis D-1, removes sulfur from DBT to form 2-hydroxybiphenyl using four enzymes, DszC, DszA, DszB, and flavin reductase. In this study, we purified and characterized the flavin reductase from R. erythropolis D-1 grown in a medium containing DBT as the sole source of sulfur. It is conceivable that the enzyme is essential for two monooxygenase (DszC and DszA) reactions in vivo. The purified flavin reductase contains no chromogenic cofactors and was found to have a molecular mass of 86 kDa and four identical 22-kDa subunits. The enzyme catalyzed NADH-dependent reduction of flavin mononucleotide (FMN), and the K m values for NADH and FMN were 208 and 10.8 M, respectively. Flavin adenine dinucleotide was a poor substrate, and NADPH was inert. The enzyme did not catalyze reduction of any nitroaromatic compound. The optimal temperature and optimal pH for enzyme activity were 35°C and 6.0, respectively, and the enzyme retained 30% of its activity after heat treatment at 80°C for 30 min. The N-terminal amino acid sequence of the purified flavin reductase was identical to that of DszD of R. erythropolis IGTS8 (K. A. Gray, O. S. Pogrebinsky, G. T. Mrachko, L. Xi, D. J. Monticello, and C. H. Squires, Nat. Biotechnol. 14:1705-1709, 1996). The flavin reductase gene was amplified with primers designed by using dszD of R. erythropolis IGTS8, and the enzyme was overexpressed in Escherichia coli. The specific activity in crude extracts of the overexpressed strain was about 275-fold that of the wild-type strain.Organic sulfur compounds are found in fossil fuels, the combustion of which causes serious environmental problems, such as acid rain. At the refinery, hydrodesulfurization is currently performed to remove sulfur compounds from fossil fuels. This process is done at high temperatures and pressures by metal catalysis and is effective for removing inorganic sulfur and simple organic sulfur compounds. However, it is difficult to remove polycyclic sulfur compounds. As legislative limits on sulfur emissions have become tighter, the need to remove polycyclic sulfur compounds from fuel has become more pressing. Dibenzothiophene (DBT) is considered a model polycyclic sulfur compound contained in fossil fuels. It has been reported that some bacteria utilize DBT as a sole source of sulfur without breaking its carbon-carbon backbone. This sulfur-specific pathway has been extensively studied by using two Rhodococcus strains, Rhodococcus erythropolis IGTS8 (7, 11, 13) and R. erythropolis D-1 (10, 19, 20). The genes encoding enzymes involved in this pathway have been cloned and sequenced in R. erythropolis IGTS8 (2, 3, 25) and the thermophilic desulfurizing bacterium Paenibacillus sp. strain A11-2 (9). In this pathway, DBT is oxidized to DBT sulfone via DBT sulfoxide by DszC, DBT sulfone is converted to 2Ј-hydroxybiphenyl 2-sulfinic acid (HBPSi) by DszA, and HBPSi is desulfurized to 2-hydroxybiphenyl by DszB (Fig. 1). Flavin reductase is necessary for monooxygenase reac...
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