Crystal structure of DszC from Rhodococcus sp. XP at 1.79 Å

The dibenzothiophene (DBT) monooxygenase DszC, which is the key initiating enzyme in “4S” metabolic pathway, catalyzes sequential sulphoxidation reaction of DBT to DBT sulfoxide (DBTO), then DBT sulfone (DBTO₂). Here, we report the crystal structure of DszC from Rhodococcus sp. XP at 1.79 Å. Intriguingly, two distinct conformations occur in the flexible lid loops adjacent to the active site (residue 280–295, between α9 and α10). They are named “open”' and “closed” state respectively, and might show the status of the free and ligand‐bound DszC. The molecular docking results suggest that the reduced FMN reacts with an oxygen molecule at C4a position of the isoalloxazine ring, producing the C4a‐(hydro)peroxyflavin intermediate which is stabilized by H391 and S163. H391 may contribute to the formation of the C4a‐(hydro)peroxyflavin by acting as a proton donor to the proximal peroxy oxygen, and it might also be involved in the protonation process of the C4a‐(hydro)xyflavin. Site‐directed mutagenesis study shows that mutations in the residues involved either in catalysis or in flavin or substrate‐binding result in a complete loss of enzyme activity, suggesting that the accurate positions of flavin and substrate are crucial for the enzyme activity. Proteins 2014; 82:1708–1720. © 2014 Wiley Periodicals, Inc.     

Purification, characterization and crystallization of enzymes for dibenzothiophene desulfurization

DszC and DszA, DBT monooxygenase and DBT sulfone monooxygenase, respectively, involved in dibenzothiophene (DBT) desulfurization, were purified to homogeneity from Rhodococcus erythropolis D-1. The two enzymes were crystallized and enzymologically characterized. We found a high activity of flavin reductase in the non-DBT-desulfurizing bacterium, Paenibacillus polymyxa A-1, which is essential for DszC and A activities, and purified to homogeneity and characterized the enzyme.     

Residue 345 of dibenzothiophene (DBT) sulfone monooxygenase is involved in C-S bond cleavage specificity of alkylated DBT sulfones

Rhodococcus erythropolis IGTS8 that possesses dibenzothiophene sulfone monooxygenase mutated at residue 345 (Q345A), can degrade octyl sulfide on which the wild strain cannot grow. Residue 345 and the neighbouring residues were changed by site-directed mutagenesis. Only DszA changed at residue 345 gave an altered C-S bond cleavage pattern of 3-methyl DBT sulfone. This residue is therefore involved in C-S bond cleavage specifically for alkylated DBT sulfone.     

Structural insights into the stabilization of active,tetrameric DszC by its C‐terminus

Dibenzothiophene (DBT) is a typical sulfur‐containing compound found in fossil fuels. This compound and its derivatives are resistant to the hydrodesulfurization method often used in industry, but they are susceptible to enzymatic desulfurization via the 4S pathway, which is a well‐studied biochemical pathway consisting of four enzymes. DBT monooxygenase (DszC) from Rhodococcus erythropolis is involved in the first step of the 4S pathway. We determined the crystal structure of DszC, which reveals that, in contrast to several homologous proteins, the C‐terminus (410–417) of DszC participates in the stabilization of the substrate‐binding pocket. Analytical ultracentrifugation analysis and enzymatic assays confirmed that the C‐terminus is important for the stabilization of the active conformation of the substrate‐binding pocket and the tetrameric state. Therefore, the C‐terminus of DszC plays a significant role in the catalytic activity of this enzyme. Proteins 2014; 82:2733–2743. © 2014 Wiley Periodicals, Inc.     

Combining co-culturing of Paenibacillus strains and Vitreoscilla hemoglobin expression as a strategy to improve biodesulfurization

Enhancement of the desulfurization activities of Paenibacillus strains 32O-W and 32O-Y were investigated using dibenzothiophene (DBT) and DBT sulfone (DBTS) as sources of sulphur in growth experiments. Strains 32O-W, 32O-Y and their co-culture (32O-W plus 32O-Y), and Vitreoscilla hemoglobin (VHb) expressing recombinant strain 32O-Yvgb and its co-culture with strain 32O-W were grown at varying concentrations (0·1–2 mmol l⁻¹) of DBT or DBTS for 96 h, and desulfurization measured by production of 2-hydroxybiphenyl (2-HBP) and disappearance of DBT or DBTS. Of the four cultures grown with DBT as sulphur source, the best growth occurred for the 32O-Yvgb plus 32O-W co-culture at 0·1 and 0·5 mmol l⁻¹ DBT. Although the presence of vgb provided no consistent advantage regarding growth on DBTS, strain 32O-W, as predicted by previous work, was shown to contain a partial 4S desulfurization pathway allowing it to metabolize this 4S pathway intermediate.     

Conversion of dibenzothiophene by the mushrooms <Emphasis Type="Italic">Agrocybe aegerita</Emphasis> and <Emphasis Type="Italic">Coprinellus radians</Emphasis> and their extracellular peroxygenases

The conversion of the heterocycle dibenzothiophene (DBT) by the agaric basidiomycetes Agrocybe aegerita and Coprinellus radians was studied in vivo and in vitro with whole cells and with purified extracellular peroxygenases, respectively. A. aegerita oxidized DBT (110 μM) by 100% within 16 days into eight different metabolites. Among the latter were mainly S-oxidation products (DBT sulfoxide, DBT sulfone) and in lower amounts, ring-hydroxylation compounds (e.g., 2-hydroxy-DBT). C. radians converted about 60% of DBT into DBT sulfoxide and DBT sulfone as the sole metabolites. In vitro tests with purified peroxygenases were performed to compare the product pattern with the metabolites formed in vivo. Using ascorbic acid as radical scavenger, a total of 19 and seven oxygenation products were detected after DBT conversion by the peroxygenases of A. aegerita (AaP) and C. radians (CrP), respectively. Whereas ring hydroxylation was favored over S-oxidation by AaP (again 2-hydroxy-DBT was identified), CrP formed DBT sulfoxide as major product. This finding suggests that fungal peroxygenases can considerably differ in their catalytic properties. Using H₂ ¹⁸O₂, the origin of oxygen was proved to be the peroxide. Based on these results, we propose that extracellular peroxygenases may be involved in the oxidation of heterocycles by fungi also under natural conditions.     

Cloning and expressing DBT (dibenzothiophene) monooxygenase gene (dszC) from Rhodococcus sp. DS-3 in Escherichia coli

Dibenzothiophene (DBT) monooxygenase (DszC) catalysis, the first and also the key step in the microbial DBT desulfurization, is the conversion of DBT to DBT sulfone (DBTO₂). In this study, dszC of a DBT-desulfurizing bacterium Rhodococcus sp. DS-3 was cloned by PCR. The sequence cloned was 99% homologous to Rhodococcus erythropolis IGTS8 that was reported in the Genebank. The gene dszC could be overexpressed effectively after being inserted into plasmid pET28a and transformed into E. coli BL21 strain. The expression amount of DszC was about 20% of total supernatant at low temperature. The soluble DszC in the supernatant was purified by Ni²⁺ chelating His-Tag resin column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to electronics purity. Only one band was detected by Western-blotting, which is for the antibody released in mouse against purified DszC in the expression product of BL21 (DE3, paC5) and Rhodococcus sp. DS-3. The activity of purified DszC was 0.36 U. DszC can utilize the organic compound such as DBT and methyl-DBT, but not DBT derivates such as DBF, which has no sulfur or inorganic sulfur. __________ Translated from Acta Scientiarum Naturalium Universitatis Nankaiensis, 2005, 38(6): 1–6 [译自: 南开大学学报 (自然科学版), 2005, 38(6): 1–6]     

Developments in destructive and non-destructive pathways for selective desulfurizations in oil-biorefining processes

Biocatalytic desulfurization is still not a commercial technology, but conceptual engineering and sensitivity analyses have shown that the approach is very promising. The purpose of this paper is to investigate further some aspects of the biodesulphurization pathways, discussing the non-destructive pathway with the well-known Rhodococcus rhodochrous IGTS8. Findings revealed byproducts, such as 2′-hydroxybiphenyl (HBP), sulfite and sulfate, obtained by the desulfurization of dibenzothiophene (DBT), to exert an inhibiting effect. The results suggest that IGTS8 may follow two different metabolic pathways in stationary-growth-phase cells or under growing conditions. The first pathway is characterized by oxidative steps, which convert DBT to DBT sulfoxide and to DBT sulfone. The sulfone is transformed to 2-(2′-hydroxyphenyl)benzene sulfinate and then to HBP and sulfite by a sulfinic acid hydrolase. In the second pathway the sulfone is further oxidized to 2-(2′-hydroxyphenyl)benzene sulfonate and then to HBP and sulfate by a sulfonic acid hydrolase. Experiments using benzene sulfonic acid suggest that the sulfonic acid hydrolase is an induced enzyme. Received: 8 June 1998 / Received revision: 1 October 1998 / Accepted: 2 October 1998     

Cloning and expressing DBT (dibenzothiophene) monooxygenase gene (dszC) from Rhodococcus sp. DS-3 in Escherichia coli

Dibenzothiophene (DBT) monooxygenase (DszC)catalysis,the first and also the key step in the microbial DBT desulfurization,is the conversion of DBT to DBT sulfone (DBTO2).In this study,dszC of a DBT-desulfiaizing bacterium Rhodococcus sp.DS-3 was cloned by PCR.The sequence cloned was 99% homologous to Rhodococcus erythropolis IGTS8 that was reported in the Genebank.The gene dszC could be overexpressed effectively after being inserted into plasmid pET28a and transformed into E.coli BL21 strain.The expression amount of DszC was about 20% of total supernatant at low temperature.The soluble DszC in the supematant was purified by Ni2+ chelating His-Tag resin column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to electronics purity.Only one band was detected by Western-blotting,which is for the antibody released in mouse against purified DszC in the expression product of BL21 (DE3,paC5) and Rhodococcus sp.DS-3.The activity of purified DszC was 0.36 U.DszC can utilize the organic compound such as DBT and methyl-DBT,hut not DBT derivates such as DBF,which has no sulfur or inorganic sulfur.     

Synthesis of novel bis‐sulfone derivatives and their inhibition properties on some metabolic enzymes including carbonic anhydrase,acetylcholinesterase, and butyrylcholinesterase

In this study, a series of novel bis‐sulfone compounds ( 2a‐2j ) were synthesized by oxidation of the bis‐sulfides under mild reaction conditions. The bis‐sulfone derivatives were characterized by ¹H‐NMR, ¹³C‐NMR, Fourier‐transform infrared spectroscopy, and elemental analysis techniques. Nuclear Overhauser effect experiments were performed to determine the orientation of the sulfonyl groups in bis‐sulfone derivatives. Here, we report the synthesis and testing of novel bis‐sulfone compound–based hybrid scaffold of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors for the development of novel molecules toward the therapy of Alzheimer's disease. The novel synthesized bis‐sulfone compounds demonstrated K_i values between 11.4 ± 3.4 and 70.7 ± 23.2 nM on human carbonic anhydrase I isozyme (hCA I), 28.7 ± 6.6 to 77.6 ± 5.6 nM on human carbonic anhydrase II isozyme (hCA II), 18.7 ± 2.61 to 95.4 ± 25.52 nM on AChE, and 9.5 ± 2.1 to 95.5 ± 1.2 nM on BChE enzymes. The results showed that novel bis‐sulfone derivatives can have promising drug potential for glaucoma, leukemia, epilepsy, and Alzheimer's disease, which are associated with the high enzymatic activity of hCA I, hCA II, AChE, and BChE enzymes.     

Biodesulfurization of Naphthothiophene and Benzothiophene through Selective Cleavage of Carbon-Sulfur Bonds by Rhodococcus sp. Strain WU-K2R

Naphtho[2,1-b]thiophene (NTH) is an asymmetric structural isomer of dibenzothiophene (DBT), and in addition to DBT derivatives, NTH derivatives can also be detected in diesel oil following hydrodesulfurization treatment. Rhodococcus sp. strain WU-K2R was newly isolated from soil for its ability to grow in a medium with NTH as the sole source of sulfur, and growing cells of WU-K2R degraded 0.27 mM NTH within 7 days. WU-K2R could also grow in the medium with NTH sulfone, benzothiophene (BTH), 3-methyl-BTH, or 5-methyl-BTH as the sole source of sulfur but could not utilize DBT, DBT sulfone, or 4,6-dimethyl-DBT. On the other hand, WU-K2R did not utilize NTH or BTH as the sole source of carbon. By gas chromatography-mass spectrometry analysis, desulfurized NTH metabolites were identified as NTH sulfone, 2′-hydroxynaphthylethene, and naphtho[2,1-b]furan. Moreover, since desulfurized BTH metabolites were identified as BTH sulfone, benzo[c][1,2]oxathiin S-oxide, benzo[c][1,2]oxathiin S,S-dioxide, o-hydroxystyrene, 2-(2′-hydroxyphenyl)ethan-1-al, and benzofuran, it was concluded that WU-K2R desulfurized NTH and BTH through the sulfur-specific degradation pathways with the selective cleavage of carbon-sulfur bonds. Therefore, Rhodococcus sp. strain WU-K2R, which could preferentially desulfurize asymmetric heterocyclic sulfur compounds such as NTH and BTH through the sulfur-specific degradation pathways, is a unique desulfurizing biocatalyst showing properties different from those of DBT-desulfurizing bacteria.     

Biodesulfurization of naphthothiophene and benzothiophene through selective cleavage of carbon-sulfur bonds by Rhodococcus sp. strain WU-K2R

Naphtho[2,1-b]thiophene (NTH) is an asymmetric structural isomer of dibenzothiophene (DBT), and in addition to DBT derivatives, NTH derivatives can also be detected in diesel oil following hydrodesulfurization treatment. Rhodococcus sp. strain WU-K2R was newly isolated from soil for its ability to grow in a medium with NTH as the sole source of sulfur, and growing cells of WU-K2R degraded 0.27 mM NTH within 7 days. WU-K2R could also grow in the medium with NTH sulfone, benzothiophene (BTH), 3-methyl-BTH, or 5-methyl-BTH as the sole source of sulfur but could not utilize DBT, DBT sulfone, or 4,6-dimethyl-DBT. On the other hand, WU-K2R did not utilize NTH or BTH as the sole source of carbon. By gas chromatography-mass spectrometry analysis, desulfurized NTH metabolites were identified as NTH sulfone, 2'-hydroxynaphthylethene, and naphtho[2,1-b]furan. Moreover, since desulfurized BTH metabolites were identified as BTH sulfone, benzo[c][1,2]oxathiin S-oxide, benzo[c][1,2]oxathiin S,S-dioxide, o-hydroxystyrene, 2-(2'-hydroxyphenyl)ethan-1-al, and benzofuran, it was concluded that WU-K2R desulfurized NTH and BTH through the sulfur-specific degradation pathways with the selective cleavage of carbon-sulfur bonds. Therefore, Rhodococcus sp. strain WU-K2R, which could preferentially desulfurize asymmetric heterocyclic sulfur compounds such as NTH and BTH through the sulfur-specific degradation pathways, is a unique desulfurizing biocatalyst showing properties different from those of DBT-desulfurizing bacteria.     

Desulfurization of dibenzothiophene by Corynebacterium sp. strain SY1.

Strain SY1, identified as a Corynebacterium sp., was isolated on the basis of the ability to utilize dibenzothiophene (DBT) as a sole source of sulfur. Strain SY1 could utilize a wide range of organic and inorganic sulfur compounds, such as DBT sulfone, dimethyl sulfide, dimethyl sulfoxide, dimethyl sulfone, CS2, FeS2, and even elemental sulfur. Strain SY1 metabolized DBT to dibenzothiophene-5-oxide, DBT sulfone, and 2-hydroxybiphenyl, which was subsequently nitrated to produce at least two different hydroxynitrobiphenyls during cultivation. These metabolites were separated by silica gel column chromatography and identified by nuclear magnetic resonance, UV, and mass spectral techniques. Resting cells of SY1 desulfurized toluenesulfonic acid and released sulfite anion. On the basis of these results, a new DBT degradation pathway is proposed.     

Desulfurization of dibenzothiophene by Corynebacterium sp. strain SY1.

Strain SY1, identified as a Corynebacterium sp., was isolated on the basis of the ability to utilize dibenzothiophene (DBT) as a sole source of sulfur. Strain SY1 could utilize a wide range of organic and inorganic sulfur compounds, such as DBT sulfone, dimethyl sulfide, dimethyl sulfoxide, dimethyl sulfone, CS2, FeS2, and even elemental sulfur. Strain SY1 metabolized DBT to dibenzothiophene-5-oxide, DBT sulfone, and 2-hydroxybiphenyl, which was subsequently nitrated to produce at least two different hydroxynitrobiphenyls during cultivation. These metabolites were separated by silica gel column chromatography and identified by nuclear magnetic resonance, UV, and mass spectral techniques. Resting cells of SY1 desulfurized toluenesulfonic acid and released sulfite anion. On the basis of these results, a new DBT degradation pathway is proposed.     

Structural conservation of the B subunit in the ammonia monooxygenase/particulate methane monooxygenase superfamily

The ammonia monooxygenase (AMO)/particulate methane monooxygenase (pMMO) superfamily is a diverse group of membrane‐bound enzymes of which only pMMO has been characterized on the molecular level. The pMMO active site is believed to reside in the soluble N‐terminal region of the pmoB subunit. To understand the degree of structural conservation within this superfamily, the crystal structure of the corresponding domain of an archaeal amoB subunit from Nitrosocaldus yellowstonii has been determined to 1.8 Å resolution. The structure reveals a remarkable conservation of overall fold and copper binding site location as well as several notable differences that may have implications for function and stability. Proteins 2014; 82:2263–2267. © 2014 Wiley Periodicals, Inc.     

Selective Desulfurization of Dibenzothiophene by Rhodococcus erythropolis D-1

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 DBT-desulfurizing ability than other microorganisms reported previously.     

Biodesulfurization of dibenzothiophene by a newly isolated Rhodococcus erythropolis strain

A new dibenzothiophene (DBT) desulfurizing bacterium was isolated from oil-contaminated soils in Iran. HPLC analysis and PCR-based detection of the presence of the DBT desulfurization genes (dszA, dszB and dszC) indicate that this strain converts DBT to 2-hydroxybiphenyl (2-HBP) via the 4S pathway. The strain, identified as Rhodococcus erythropolis SHT87, can utilize DBT, dibenzothiophene sulfone, thiophene, 2-methylthiophene and dimethylsulfoxide as a sole sulfur source for growth at 30 °C.The maximum specific desulfurization activity of strain SHT87 resting cells in aqueous and biphasic organic–aqueous systems at 30 °C was determined to be 0.36 and 0.47 μmol 2-HBP min⁻¹ (g dry cell)⁻¹, respectively. Three mM DBT was completely metabolized by SHT87 resting cells in the aqueous and biphasic systems within 10 h. The rate and the extent of the desulfurization reaction by strain SHT87 suggest that this strain can be used for the biodesulfurization of diesel oils.     

Identification and functional analysis of the genes encoding dibenzothiophene-desulfurizing enzymes from thermophilic bacteria

Thermophilic bacteria Bacillus subtilis WU-S2B and Mycobacterium phlei WU-F1 desulfurize dibenzothiophene (DBT) and alkylated DBTs through specific cleavage of the carbon-sulfur bonds over a temperature range up to 52°C. In order to identify and functionally analyze the DBT-desulfurization genes, the gene cluster containing bdsA, bdsB, and bdsC was cloned from B. subtilis WU-S2B. The nucleotide and amino acid sequences of bdsABC show homologies to those of the other known DBT-desulfurization genes and enzymes; e.g. a nucleotide sequence homology of 61.0% to dszABC of the mesophilic bacterium Rhodococcus sp. IGTS8 and 57.8% to tdsABC of the thermophilic bacterium Paenibacillus sp. A11-2. Deletion and subcloning analysis of bdsABC revealed that the gene products of bdsC, bdsA and bdsB oxidized DBT to DBT sulfone (DBTO₂), converted DBTO₂ to 2-hydroxybiphenyl-2-sulfinate (HBPSi), and desulfurized HBPSi to 2-hydroxybiphenyl (2-HBP), respectively. Resting cells of a recombinant Escherichia coli JM109 harboring bdsABC converted DBT to 2-HBP over a temperature range of 30–52°C, indicating that the gene products of bdsABC were functional in the recombinant. The activities of DBT degradation at 50°C and DBT desulfurization (2-HBP production) at 40°C in resting cells of the recombinant were approximately five times and twice, respectively, as high as those in B. subtilis WU-S2B. The recombinant E. coli cells also degraded alkylated DBTs, such as 2,8-dimethylDBT and 4,6-dimethylDBT. The nucleotide sequences of B. subtilis WU-S2B bdsABC and the corresponding genes from M. phlei WU-F1 were found to be completely identical to each other although the strains are genetically different.     

Crystal structure of yeast nitronate monooxygenase from Cyberlindnera saturnus

Nitronate monooxygenase (NMO) is an FMN‐dependent enzyme that oxidizes the neurotoxin propionate 3‐nitronate (P3N) and represents the best‐known system for P3N detoxification in different organisms. The crystal structure of the first eukaryotic Class I NMO from Cyberlindnera saturnus (CsNMO) has been solved at 1.65 Å resolution and refined to an R‐factor of 14.0%. The three‐dimensional structures of yeast CsNMO and bacterial PaNMO are highly conserved with the exception of three additional loops on the surface in the CsNMO enzyme and differences in four active sites residues. A PEG molecule was identified in the structure and formed extensive interactions with CsNMO, suggesting a specific binding site; however, 8% PEG showed no significant effect on the enzyme activity. This new crystal structure of a eukaryotic NMO provides insight into the function of this class of enzymes.     

Thermophilic Biodesulfurization of Various Heterocyclic Sulfur Compounds and Crude Straight-Run Light Gas Oil Fraction by a Newly Isolated Strain Mycobacterium phlei WU-0103

Various heterocyclic sulfur compounds such as naphtho[2,1-b]thiophene (NTH) and benzo[b]thiophene (BTH) derivatives can be detected in diesel oil, in addition to dibenzothiophene (DBT) derivatives. Mycobacterium phlei WU-0103 was newly isolated as a bacterial strain capable of growing in a medium with NTH as the sulfur source at 50°C. M. phlei WU-0103 could degrade various heterocyclic sulfur compounds, not only NTH and its derivatives but also DBT, BTH, and their derivatives at 45°C. When M. phlei WU-0103 was cultivated with the heterocyclic sulfur compounds such as NTH, NTH 3,3-dioxide, DBT, BTH, and 4,6-dialkylDBTs as sulfur sources, monohydroxy compounds and sulfone compounds corresponding to starting heterocyclic sulfur compounds were detected by gas chromatography–mass spectrometry analysis, suggesting the sulfur-specific desulfurization pathways for heterocyclic sulfur compounds. Moreover, total sulfur content in 12-fold-diluted crude straight-run light gas oil fraction was reduced from 1000 to 475 ppm S, with 52% reduction, by the biodesulfurization treatment at 45°C with growing cells of M. phlei WU-0103. Gas chromatography analysis with a flame photometric detector revealed that most of the resolvable peaks, such as those corresponding to alkylated derivatives of NTH, DBT, and BTH, disappeared after the biodesulfurization treatment. These results indicated that M. phlei WU-0103 may have a good potential as a biocatalyst for practical biodesulfurization of diesel oil.