Crystal structure of dibenzothiophene sulfone monooxygenase BdsA from Bacillus subtilis WU-S2B.

The dibenzothiophene (DBT) sulfone monooxygenase BdsA from Bacillus subtilis WU-S2B catalyzes the conversion of DBT sulfone to 2'-hydroxybiphenyl 2-sulfinate. We report the crystal structures of BdsA at a resolution of 2.80 Å. BdsA exists as a homotetramer with a dimer-of-dimers configuration in the crystal, and the interaction between E288 and R296 in BdsA is important for tetramer formation. A structural comparison with homologous proteins shows that the orientation and location of the α9-α12 helices in BdsA are closer to those of the closed form than those of the open form in the EDTA monooxygenase EmoA. Proteins 2017; 85:1171-1177. © 2017 Wiley Periodicals, Inc.

Crystal structures of apo-DszC and FMN-bound DszC from Rhodococcus erythropolis D-1.

UNLABELLED: 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: 3X0X) and DszC-FMN (PDB code: 3X0Y) have been deposited in the Protein Data Bank (http://www.rcsb.org).

Detection and characterization of a thermophilic biotin biosynthetic enzyme, 7-keto-8-aminopelargonic acid synthase, from various thermophiles.

By detailed BLAST searches of the genome database of various thermophiles, five ORFs with similarity to the bioF gene, which encodes 7-keto-8-aminopelargonic acid synthase (BioF) involved in biotin biosynthesis, of Escherichia coli were found: AqbioF, CltbioF, GkbioF, SytbioF, and TsebioF, from Aquifex aeolicus VF5, Clostridium thermocellum ATCC27405, Geobacillus kaustophilus JCM12893, Symbiobacterium thermophilum IAM14863, and Thermosynechococcus elongatus BP-1 respectively. The five purified recombinant bioF gene products, which were overexpressed in E. coli, had the enzyme activity of BioF. The optimum temperature range and thermostability of five BioFs, AqBioF, CltBioF, GkBioF, SytBioF, and TseBioF, were higher than those of E. coli BioF. In particular, AqBioF was found to show the highest thermostability of the α-oxoamine synthase family enzymes reported to date. Substrate specificity experiments revealed that SytBioF was also able to catalyze the reaction of 2-amino-3-ketobutyrate CoA ligase, a member of the α-oxoamine synthase family, and that it used acetyl-CoA and glycine as substrates, like the TTHA1582 protein of Thermus thermophilus. The other purified BioFs, AqBioF and GkBioF, did not show any activity with acyl-CoAs and amino acids other than pimeloyl-CoA and L-alanine as substrates.

Isolation and characterization of a novel fucoidan-degrading microorganism.

A bacterium utilizing fucoidan from the brown alga Cladosiphon okamuranus as sole carbon source was isolated and identified as Flavobacterium sp. F-31. The strain produced intracellular enzymes involved in fucoidan degradation and desulfation, but desulfation activity was not detected until the molecular weight of fucoidan fell to less than several tens of thousands due to enzymatic degradation. Only fucoidan proved to be an inducible substance for the production of the degrading enzymes.

Novel reactivity of dibenzothiophene monooxygenase from Bacillus subtilis WU-S2B.

Dibenzothiophene monooxygenase (BdsC) from Bacillus subtilis WU-S2B utilized aromatic compounds not having sulfur atoms as substrates. It acted on indole and its derivatives to form indigoid pigments, and also utilized indoline and phenoxazine. In addition, BdsC exhibited activity toward benzothiophene (BT) derivatives but not BT, suggesting that it shows wide reactivity toward aromatic compounds.

The first thermophilic alpha-oxoamine synthase family enzyme that has activities of 2-amino-3-ketobutyrate CoA ligase and 7-keto-8-aminopelargonic acid synthase: cloning and overexpression of the gene from an extreme thermophile, Thermus thermophilus, and characterization of its gene product.

The first thermophilic alpha-oxoamine synthase family enzyme was identified. The gene (ORF TTHA1582), which is annotated to code putative alpha-oxoamine synthase family enzymes, 7-keto-8-aminopelargonic acid (KAPA) synthase (BioF, 8-amino-7-oxononanoate synthase, EC 2.3.1.47) and 2-amino-3-ketobutyrate CoA ligase (KBL, EC 2.3.1.29), in a genomic database, was cloned from an extreme thermophile, Thermus thermophilus, and overexpressed in Escherichia coli. The recombinant TTHA1582 protein was purified and characterized. It exhibited activity of BioF, which catalyzes the condensation of pimeloyl-CoA and L-alanine to produce a biotin intermediate KAPA, CoASH, and CO(2) with pyridoxal 5'-phosphate as a cofactor. The protein is a dimer with a subunit of 43 kDa that shows an amino acid sequence identity of 35% with E. coli BioF. The optimum temperature and pH were about 70 degrees C and about 6.0. The enzyme showed high thermostability at temperatures of up to 70 degrees C for 1 h, and a half-life of 1 h at 80 degrees C. Thus the TTHA1582 protein was found to have the highest optimum temperature and thermostablility of the alpha-oxoamine synthase family enzymes so far reported. Substrate specificity experiments revealed that it was also able to catalyze the KBL reaction, which used acetyl-CoA and glycine as substrates, and that enzyme activity was seen with the following combinations of substrates: acetyl-CoA and glycine, L-alanine, or L-serine; pimeloyl-CoA and L-alanine, glycine, or L-serine; palmitoyl-CoA and L-alanine. This suggests that the recombinant TTHA1582 protein has broad substrate specificity, unlike the reported mesophilic enzymes of the alpha-oxoamine synthase family.

Improvement of 2'-hydroxybiphenyl-2-sulfinate desulfinase, an enzyme involved in the dibenzothiophene desulfurization pathway, from Rhodococcus erythropolis KA2-5-1 by site-directed mutagenesis.

In the microbial dibenzothiophene desulfurization pathway, 2'-hydroxybiphenyl-2-sulfinate is converted to 2-hydroxybiphenyl and sulfinate by desulfinase (DszB) at the last step, and this reaction is rate-limiting for the whole pathway. The catalytic activity and thermostability of DszB were enhanced by the two amino acid substitutions. Based on information on the 3-D structure of DszB and a comparison of amino acid sequences between DszB and reported thermophilic and thermostable homologs (TdsB and BdsB), two amino acid residues, Tyr63 and Gln65, were selected as targets to mutate and improve DszB. These two residues were replaced by several amino acids, and the promising mutant enzymes were purified and their properties were examined. Among the wild-type and mutant enzymes, Y63F had higher catalytic activity but similar thermostability, and Q65H showed higher thermostability but less catalytic activity and affinity for the substrate. To compensate for these drawbacks, the double mutant enzyme Y63F-Q65H was purified and its properties were investigated. This mutant enzyme showed higher thermostability without loss of catalytic activity or affinity for the substrate. These superior properties of the mutant enzyme have also been confirmed with resting cells harboring the mutant gene.

Crystal structure and desulfurization mechanism of 2'-hydroxybiphenyl-2-sulfinic acid desulfinase.

The desulfurization of dibenzothiophene in Rhodococcus erythropolis is catalyzed by two monooxygenases, DszA and DszC, and a desulfinase, DszB. In the last step of this pathway, DszB hydrolyzes 2'-hydroxybiphenyl-2-sulfinic acid into 2-hydroxybiphenyl and sulfite. We report on the crystal structures of DszB and an inactive mutant of DszB in complex with substrates at resolutions of 1.8A or better. The overall fold of DszB is similar to those of periplasmic substrate-binding proteins. In the substrate complexes, biphenyl rings of substrates are recognized by extensive hydrophobic interactions with the active site residues. Binding of substrates accompanies structural changes of the active site loops and recruits His(60) to the active site. The sulfinate group of bound substrates forms hydrogen bonds with side chains of Ser(27), His(60), and Arg(70), each of which is shown by site-directed mutagenesis to be essential for the activity. In our proposed reaction mechanism, Cys(27) functions as a nucleophile and seems to be activated by the sulfinate group of substrates, whereas His(60) and Arg(70) orient the syn orbital of sulfinate oxygen to the sulfhydryl hydrogen of Cys(27) and stabilize the negatively charged reaction intermediate. Cys, His, and Arg residues are conserved in putative proteins homologous to DszB, which are presumed to constitute a new family of desulfinases.

Reaction of serine-glyoxylate aminotransferase with the alternative substrate ketomalonate indicates rate-limiting protonation of a quinonoid intermediate.

Serine-glyoxylate aminotransferase (SGAT) from Hyphomicrobium methylovorum is a pyridoxal 5'-phosphate (PLP) enzyme that catalyzes the interconversion of L-serine and glyoxylate to hydroxypyruvate and glycine. The primary deuterium isotope effect using L-serine 2-D is one on (V/K)serine and V in the steady state. Pre-steady-state experiments also indicate that there is no primary deuterium isotope effect with L-serine 2-D. The results suggest there is no rate limitation by abstraction of the alpha proton of L-serine in the SGAT reaction. In the steady-state a solvent deuterium isotope effect of about 2 was measured on (V/K)L-serine and (V/K)ketomalonate and about 5.5 on V. Similar solvent isotope effects were observed in the pre-steady-state for the natural substrates and the alternative substrate ketomalonate. In the pre-steady-state, no reaction intermediates typical of PLP enzymes were observed with the substrates L-serine, glyoxylate, and hydroxypyruvate. The data suggest that breakdown and formation of the ketimine intermediate is the primary rate-limiting step with the natural substrates. In contrast, using the alternative substrate ketomalonate, pre-steady-state experiments display the transient formation of a 490 nm absorbing species typical of a quinonoid intermediate. The solvent isotope effect results also suggest that with ketomalonate as substrate protonation at C(alpha) is the slowest step in the SGAT reaction. This is the first report of a rate-limiting protonation of a quinonoid at C(alpha) of the external Schiff base in an aminotransferase reaction.

Dibenzothiophene desulfurizing enzymes from moderately thermophilic bacterium Bacillus subtilis WU-S2B: purification, characterization and overexpression.

The moderately thermophilic bacterium Bacillus subtilis WU-S2B desulfurized dibenzothiophene (DBT) at 50 degrees C through the selective cleavage of carbon-sulfur bonds. In this study, three enzymes involved in the microbial DBT desulfurization were purified and characterized. The first two enzymes, DBT monooxygenase (BdsC) and DBT sulfone monooxygenase (BdsA), were purified from the wild-type strain, and the last one, 2'-hydroxybiphenyl 2-sulfinic acid desulfinase (BdsB), was purified from the recombinant Escherichia coli overexpressing the gene, bdsB, with chaperonin genes, groEL/ES. The genes of BdsC and BdsA were also overexpressed. The molecular weights of BdsC and BdsA were determined to be 200 and 174 kDa, respectively, by gel filtration chromatography, suggesting that both enzymes had four identical subunits. BdsB had a monomeric structure of 40 kDa. The three enzymes were characterized and compared with the corresponding enzymes (DszC, DszA, and DszB) of mesophilic desulfurization bacteria. The specific activities of BdsC, BdsA, and BdsB were 84.2, 855, and 280 units/mg, respectively, and the latter two activities were higher than those of DszA and DszB. The heat stability and optimum temperature of BdsC, BdsA, and BdsB were higher than those of DszC, DszA, and DszB. Other enzymatic properties were investigated in detail.

Enhancing effect of calcium and vanadium ions on thermal stability of bromoperoxidase from Corallina pilulifera.

Bromoperoxidase from the macro-alga Corallina pilulifera is an enzyme that possesses vanadate in the catalytic center, and shows a significant thermostability and stability toward organic solvents. The structural analysis of the recombinant enzyme overexpressed in yeast revealed that it contains one calcium atom per subunit. This has been confirmed by inductively coupled plasma emission spectrometry experiments. The study of the effect of metal ions on the apo-enzyme stability has shown that the calcium ion significantly increased the enzyme stability. In addition, vanadate also increased the thermostability and strontium and magnesium ions had similar effects as calcium. The holo-enzyme shows high stability in a range of organic solvents. The effect of the different ions and solvents on the structure of the enzyme has been studied by circular dichroism experiments. The high stability of the enzyme in the presence of organic solvents is useful for its application as a biocatalyst.

Functional analysis of two solanesyl diphosphate synthases from Arabidopsis thaliana.

Solanesyl diphosphate (SPP) is regarded as the precursor of the side-chains of both plastoquinone and ubiquinone in Arabidopsis thaliana. We previously analyzed A. thaliana SPP synthase (At-SPS1) (Hirooka et al., Biochem. J., 370, 679-686 (2003)). In this study, we cloned a second SPP synthase (At-SPS2) gene from A. thaliana and characterized the recombinant protein. Kinetic analysis indicated that At-SPS2 prefers geranylgeranyl diphosphate to farnesyl diphosphate as the allylic substrate. Several of its features, including the substrate preference, were similar to those of At-SPS1. These data indicate that At-SPS1 and At-SPS2 share their basic catalytic machinery. Moreover, analysis of the subcellular localization by the transient expression of green fluorescent protein-fusion proteins showed that At-SPS2 is transported into chloroplasts, whereas At-SPS1 is likely to be localized in the endoplasmic reticulum in the A. thaliana cells. It is known that the ubiquinone side-chain originates from isopentenyl diphosphate derived from the cytosolic mevalonate pathway, while the plastoquinone side-chain is synthesized from isopentenyl diphosphate derived from the plastidial methylerythritol phosphate pathway. Based on this information, we propose that At-SPS1 contributes to the biosynthesis of the ubiquinone side-chain and that At-SPS2 supplies the precursor of the plastoquinone side-chain in A. thaliana.

Crystallization and preliminary X-ray analyses of desulfurization enzyme DszB and its C27S mutant complexed with biphenyl-2-sulfinic acid.

DszB is a hydrolase involved in the biodegradation of dibenzothiophene in the soil bacterium Rhodococcus sp. IGTS8. DszB catalyzes the hydrolysis of 2'-hydroxybiphenyl-2-sulfinic acid to sulfite and biphenyl-2-ol. DszB and DszB C27S mutant complexed with biphenyl-2-sulfinic acid were crystallized and preliminary X-ray crystallographic analyses were conducted. The crystals of DszB were found to belong to the orthorhombic P2(1)2(1)2(1) space group, with unit-cell parameters a = 36.7, b = 82.6, c = 139.6 A, and to contain one molecule of DszB in the asymmetric unit. Crystals of DszB C27S complexed with biphenyl-2-sulfinic acid belong to space group C2, with unit-cell parameters a = 153.4, b = 45.9, c = 112.9 A, beta = 115.93 degrees. The calculated Matthews coefficient V(M) for the C2 crystals is approximately 2.3 A(3) Da(-1) if two molecules of DszB are present in the asymmetric unit.

Thermostable flavin reductase that couples with dibenzothiophene monooxygenase, from thermophilic Bacillus sp. DSM411: purification, characterization, and gene cloning.

Flavin reductase is essential for the oxygenases involved in microbial dibenzothiophene (DBT) desulfurization. An enzyme of the thermophilic strain, Bacillus sp. DSM411, was selected to couple with DBT monooxygenase (DszC) from Rhodococcus erythropolis D-1. The flavin reductase was purified to homogeneity from Bacillus sp. DSM411, and the native enzyme was a monomer of M(r) 16 kDa. Although the best substrates were flavin mononucleotide and NADH, the enzyme also used other flavin compounds and acted slightly on nitroaromatic compounds and NADPH. The purified enzyme coupled with DszC and had a ferric reductase activity. Among the flavin reductases so far characterized, the present enzyme is the most thermophilic and thermostable. The gene coded for a protein of 155 amino acids with a calculated mass of 17,325 Da. The enzyme was overproduced in Escherichia coli, and the specific activity in the crude extracts was about 440-fold higher than that of the wild-type strain, Bacillus sp. DSM411.

Modification of halogen specificity of a vanadium-dependent bromoperoxidase.

The halide specificity of vanadium-dependent bromoperoxidase (BPO) from the marine algae, Corallina pilulifera, has been changed by a single amino acid substitution. The residue R397 has been substituted by the other 19 amino acids. The mutant enzymes R397W and R397F showed significant chloroperoxidase (CPO) activity as well as BPO activity. These mutant enzymes were purified and their properties were investigated. The maximal velocities of CPO activities of the R397W and R397F enzymes were 31.2 and 39.2 units/mg, and the K(m) values for Cl(-) were 780 mM and 670 mM, respectively. Unlike the native enzyme, both mutant enzymes were inhibited by NaN(3). In the case of the R397W enzyme, the incorporation rate of vanadate into the active site was low, compared with the R397F and the wild-type enzyme. These results supported the existence of a specific halogen binding site within the catalytic cleft of vanadium haloperoxidases.

Biosynthesis of epsilon-poly-L-lysine in a cell-free system of Streptomyces albulus.

epsilon-Poly-L-lysine (epsilon-PL) is a homo-poly-amino acid characterized by a peptide bond between carboxyl and epsilon-amino groups of L-lysine. Here we report the cell-free synthesis of epsilon-PL by a sensitive radioisotopic epsilon-PL assay system. In vitro epsilon-PL synthesis depended on ATP and was not affected by ribonuclease, kanamycin, or chloramphenicol. epsilon-PL synthesizing activity was detected in the membrane fraction. The reaction product, epsilon-PL, from L-lysine was identified by MALDI-TOF MS and the number of lysine residues of the epsilon-PL products was apparently 11-34. These results suggest that the biosynthesis of epsilon-PL is nonribosomal peptide synthesis and is catalyzed by membrane bound enzyme(s). The enzyme preparation showing the epsilon-PL synthesizing activity also catalyzed lysine-dependent AMP production and an ATP-PPi exchange reaction, suggesting that L-lysine is adenylated in the first step of epsilon-PL biosynthesis.

Expression of the vanadium-dependent bromoperoxidase gene from a marine macro-alga Corallina pilulifera in Saccharomyces cerevisiae and characterization of the recombinant enzyme.

The vanadium-dependent bromoperoxidase from the marine macro-alga Corallina pilulifera was heterologously expressed in Saccharomyces cerevisiae. The enzyme was purified and crystals in "tear drop" form were obtained. The catalytic properties of the recombinant enzyme were studied and compared with those of the native enzyme purified from C. pilulifera. Differences in thermal stability and chloroperoxidase activity were observed. The recombinant enzyme retained full activity after preincubation at 65 degrees C for 20 min, but the native enzyme was completely inactivated under the same conditions. The chlorinating activity of the native enzyme was more than ten times higher than that of the recombinant enzyme. Other properties, such as K(m) values for KBr and H(2)O(2), and optimal temperature and pH, were similar for each source of C. pilulifera bromoperoxidase.

A novel enzyme, 2'-hydroxybiphenyl-2-sulfinate desulfinase (DszB), from a dibenzothiophene-desulfurizing bacterium Rhodococcus erythropolis KA2-5-1: gene overexpression and enzyme characterization.

Dibenzothiophene (DBT), a model of organic sulfur compound in petroleum, is microbially desulfurized to 2-hydroxybiphenyl (2-HBP), and the gene operon dszABC was required for DBT desulfurization. The final step in the microbial DBT desulfurization is the conversion of 2'-hydroxybiphenyl-2-sulfinate (HBPSi) to 2-HBP catalyzed by DszB. In this study, DszB of a DBT-desulfurizing bacterium Rhodococcus erythropolis KA2-5-1 was overproduced in Escherichia coli by coexpression with chaperonin genes, groEL/groES, at 25 degrees C. The recombinant DszB was purified to homogeneity and characterized. The optimal temperature and pH for DszB activity were 35 degrees C and about 7.5, respectively. The K(m) and k(cat) values for HBPSi were 8.2 microM and 0.123.s(-1), respectively. DszB has only one cysteine residue, and the mutant enzyme completely lost the activity when the cysteine residue was changed to a serine residue. This result together with experiments using inhibitors showed that the cysteine residue contributes to the enzyme activity. DszB was also inhibited by a reaction product, 2-HBP (K(i)=0.25 mM), and its derivatives, but not by the other reaction product, sulfite. The enzyme showed a narrow substrate specificity: only 2-phenylbenzene sulfinate except HBPSi served as a substrate among the aromatic and aliphatic sulfinates or sulfonates tested. DszB was thought to be a novel enzyme (HBPSi desulfinase) in that it could specifically cleave the carbon-sulfur bond of HBPSi to give 2-HBP and sulfite ion without the aid of any other proteinic components and coenzymes.

Enhancement and stabilization of desulfurization activity of Rhodococcus erythropolis KA2-5-1 by feeding ethanol and sulfur components.

We developed a fed-batch culture system fed with ethanol and restricted amounts of sulfur compounds to enhance and stabilize the desulfurizing activity in bacterial cells. In this system using dibenzothiophene (DBT) as the sole sulfur source, a desulfurizing bacterium Rhodococcus erythropolis KA2-5-1 cultivated with small amounts of sulfur showed stable desulfurizing activity and a low rate of growth. However, the cells cultured with excess amounts of sulfur showed unstable activity and a high growth rate. DBT had disadvantages as a sulfur source for cultivation because it is immiscible with water and toxic to cells. We then investigated water-soluble sulfur compounds for use as the sole sulfur source for the cultivation of R. erythropolis KA2-5-1 with desulfurizing activity, and found 2-aminoethanesulfonic acid to be the most effective. When 2-aminoethanesulfonic acid was used instead of DBT as the sole sulfur source in the fed-batch fermentation system, R. erythropolis KA2-5-1 showed the highest desulfurizing activity, 111 mmol of 2-HBP/kg-cells/h, a high growth rate (mu = 0.37/h) and a final cell concentration of 20.0 g-dry cells/l during 89 h of cultivation. The production levels of the desulfurizing enzymes in the bacterial cells cultivated with DBT or 2-aminoethanesulfonic acid were evaluated by immunoblot analysis with specific antisera, indicating that the same quantity of desulfurizing enzymes was expressed in both cases.

Enantioselective Sulfoxidation Catalyzed by Vanadium Haloperoxidases.

Vanadium haloperoxidases catalyze the oxidation of halides by hydrogen peroxide to produce hypohalous acid. We demonstrate that these enzymes also slowly mediate the enantioselective oxidation of organic sulfides (methyl phenyl sulfide, methyl p-tolyl sulfide, and 1-methoxy-4 (methylthio)benzene) to the corresponding sulfoxides (turnover frequency 1 min(-)(1)). The vanadium bromoperoxidase from the brown seaweed Ascophyllum nodosum converts methyl phenyl sulfide to the (R)-enantiomer of the sulfoxide (55% yield and 85% enantiomeric excess (ee)). At low peroxide concentrations a selectivity of 91% can be attained. The enzyme catalyzes the selective sulfoxidation reaction over a broad pH range with an optimum around pH 5-6 and remains completely functional during the reaction. When the vanadium bromoperoxidase from the red seaweed Corallina pilulifera is used the (S)-enantiomer (18% yield and 55% ee) is formed. In contrast, the vanadium chloroperoxidase from the fungus Curvularia inaequalis catalyzes the production of a racemic mixture (54% yield), which seems to be an intrinsic characteristic of this enzyme.