The Experts below are selected from a list of 723 Experts worldwide ranked by ideXlab platform
Andreas Schmid - One of the best experts on this subject based on the ideXlab platform.
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biocatalytic production of catechols using a high pressure tube in tube segmented flow microreactor
Organic Process Research & Development, 2014Co-Authors: Bartlomiej Tomaszewski, Andreas Schmid, Katja BuehlerAbstract:This study reports the synthesis of 3-phenylcatechol at the preparative scale using a continuous segmented flow tube-in-tube reactor (TiTR). 2-Hydroxybiphenyl 3-monooxygenase (HbpA) was applied as a biocatalyst for the hydroxylation reaction, which is dependent on the substrate 2-Hydroxybiphenyl, NADH, and oxygen. While the regeneration of the cofactor NADH was guaranteed by formate dehydrogenase (FDH), oxygen was supplied via the membrane surface from the outside of the reactor system. The oxygen transfer rate through the membrane of the TiTR was determined to be 24 μmol O2 min–1 mL–1 emphasizing the potential of the TiTR as promising technology for realizing gas-dependent enzymatic reactions. Residence time and total turnover number have been identified as key limiting parameters. It was possible to scale-up this system by extending the TiTR by additional residence time units. This allowed synthesis of 1 g of 3-phenylcatechol at a high space time yield of 14.5 g L–1 h–1.
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regioselective biocatalytic aromatic hydroxylation in a gas liquid multiphase tube in tube reactor
Chemcatchem, 2014Co-Authors: Bartlomiej Tomaszewski, Katja Buehler, Richard C Lloyd, Antony J Warr, Andreas SchmidAbstract:Microreactors provide higher mass transfer rates than do conventional batch reactors. A tube-in-tube microreactor was used for the NADH-dependent in vitro conversion of 2-Hydroxybiphenyl to 3-phenylcatechol that was catalysed by 2-Hydroxybiphenyl 3-monooxygenase. A biphasic reaction system allowed high substrate loadings, whereas the microreactor ensured excellent mass transfer rates between the organic and aqueous phases. Oxygen was supplied continuously by membrane aeration across the whole reaction compartment. The productivities achieved in the tube-in-tube microreactor were 38 times higher than those in previously described batch reactors and almost 4 times higher than for the same reaction in a microreactor in which aqueous, organic, and air phases were delivered through consecutive segments. This set-up is a promising concept for oxygen-dependent biocatalytic reactions in microreactors and is developing as a basis for applications in gram-scale organic biosyntheses.
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Biocatalytic Production of Catechols Using a High Pressure Tube-in-Tube Segmented Flow Microreactor
2014Co-Authors: Bartłomiej Tomaszewski, Andreas Schmid, Katja BuehlerAbstract:This study reports the synthesis of 3-phenylcatechol at the preparative scale using a continuous segmented flow tube-in-tube reactor (TiTR). 2-Hydroxybiphenyl 3-monooxygenase (HbpA) was applied as a biocatalyst for the hydroxylation reaction, which is dependent on the substrate 2-Hydroxybiphenyl, NADH, and oxygen. While the regeneration of the cofactor NADH was guaranteed by formate dehydrogenase (FDH), oxygen was supplied via the membrane surface from the outside of the reactor system. The oxygen transfer rate through the membrane of the TiTR was determined to be 24 μmol O2 min–1 mL–1 emphasizing the potential of the TiTR as promising technology for realizing gas-dependent enzymatic reactions. Residence time and total turnover number have been identified as key limiting parameters. It was possible to scale-up this system by extending the TiTR by additional residence time units. This allowed synthesis of 1 g of 3-phenylcatechol at a high space time yield of 14.5 g L–1 h–1
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bioorganometallic chemistry biocatalytic oxidation reactions with biomimetic nad nadh co factors and cp rh bpy h for selective organic synthesis
Journal of Organometallic Chemistry, 2004Co-Authors: Jochen Lutz, Richard H Fish, Frank Hollmann, Adrian Schnyder, Andreas SchmidAbstract:The biocatalytic, regioselective hydroxylation of 2-Hydroxybiphenyl to the corresponding catechol was accomplished utilizing the monooxygenase 2-Hydroxybiphenyl 3-monooxygenase (HbpA). The necessary natural nicotinamide adenine dinucleotide (NAD{sup +}) co-factor for this biocatalytic process was replaced by a biomimetic co-factor, N-benzylnicotinamide bromide, 1a. The interaction between the flavin (FAD) containing HbpA enzyme and the corresponding biomimetic NADH compound, N-benzyl-1,4-dihdronicotinamide, 1b, for hydride transfers, was shown to readily occur. The in situ recycling of the reduced NADH biomimic 1b from 1a was accomplished with [Cp*Rh(bpy)H](Cl); however, productive coupling of this regeneration reaction to the enzymatic hydroxylation reaction was not totally successful, due to a deactivation process concerning the HbpA enzyme peripheral groups; i.e., -SH or -NH{sub 2} possibly reacting with the precatalyst, [Cp*Rh(bpy)(H{sub 2}O)](Cl){sub 2}, and thus inhibiting the co-factor regeneration process. The deactivation mechanism was studied, and a promising strategy of derivatizing these peripheral -SH or -NH{sub 2} groups with a polymer containing epoxide was successful in circumventing the undesired interaction between HbpA and the precatalyst. This latter strategy allowed tandem co-factor regeneration using 1a or 2a, [Cp*Rh(bpy)(H2O)](Cl){sub 2}, and formate ion, in conjunction with the polymer bound, FAD containing HbpA enzyme to provide the catechol product.
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Bioorganometallic chemistry: biocatalytic oxidation reactions with biomimetic nad+/nadh co-factors and [cp*rh(bpy)h]+ for selective organic synthesis
Journal of Organometallic Chemistry, 2004Co-Authors: Jochen Lutz, Richard H Fish, Frank Hollmann, Adrian Schnyder, Andreas SchmidAbstract:The biocatalytic, regioselective hydroxylation of 2-Hydroxybiphenyl to the corresponding catechol was accomplished utilizing the monooxygenase 2-Hydroxybiphenyl 3-monooxygenase (HbpA). The necessary natural nicotinamide adenine dinucleotide (NAD{sup +}) co-factor for this biocatalytic process was replaced by a biomimetic co-factor, N-benzylnicotinamide bromide, 1a. The interaction between the flavin (FAD) containing HbpA enzyme and the corresponding biomimetic NADH compound, N-benzyl-1,4-dihdronicotinamide, 1b, for hydride transfers, was shown to readily occur. The in situ recycling of the reduced NADH biomimic 1b from 1a was accomplished with [Cp*Rh(bpy)H](Cl); however, productive coupling of this regeneration reaction to the enzymatic hydroxylation reaction was not totally successful, due to a deactivation process concerning the HbpA enzyme peripheral groups; i.e., -SH or -NH{sub 2} possibly reacting with the precatalyst, [Cp*Rh(bpy)(H{sub 2}O)](Cl){sub 2}, and thus inhibiting the co-factor regeneration process. The deactivation mechanism was studied, and a promising strategy of derivatizing these peripheral -SH or -NH{sub 2} groups with a polymer containing epoxide was successful in circumventing the undesired interaction between HbpA and the precatalyst. This latter strategy allowed tandem co-factor regeneration using 1a or 2a, [Cp*Rh(bpy)(H2O)](Cl){sub 2}, and formate ion, in conjunction with the polymer bound, FAD containing HbpA enzyme to provide the catechol product.
Yoshikazu Izumi - One of the best experts on this subject based on the ideXlab platform.
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crystal structures of apo dszc and fmn bound dszc from rhodococcus erythropolis d 1
FEBS Journal, 2015Co-Authors: Lijun Guan, Yoshikazu Izumi, Takashi Ohshiro, Woo Cheol Lee, Shipeng Wang, Jun Ohtsuka, Masaru TanokuraAbstract: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).
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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
Bioscience Biotechnology and Biochemistry, 2007Co-Authors: Takashi Ohshiro, Woo Cheol Lee, Masaru Tanokura, Ryo Ohkita, Takeshi Takikawa, Masanori Manabe, Yoshikazu IzumiAbstract: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.
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crystal structure and desulfurization mechanism of 2 Hydroxybiphenyl 2 sulfinic acid desulfinase
Journal of Biological Chemistry, 2006Co-Authors: Woo Cheol Lee, Yoshikazu Izumi, Takashi Ohshiro, Toshiyuki Matsubara, Masaru TanokuraAbstract: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 His60 to the active site. The sulfinate group of bound substrates forms hydrogen bonds with side chains of Ser27, His60, and Arg70, each of which is shown by site-directed mutagenesis to be essential for the activity. In our proposed reaction mechanism, Cys27 functions as a nucleophile and seems to be activated by the sulfinate group of substrates, whereas His60 and Arg70 orient the syn orbital of sulfinate oxygen to the sulfhydryl hydrogen of Cys27 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.
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crystallization and preliminary x ray analyses of desulfurization enzyme dszb and its c27s mutant complexed with biphenyl 2 sulfinic acid
Acta Crystallographica Section D-biological Crystallography, 2004Co-Authors: Woo Cheol Lee, Yoshikazu Izumi, Takashi Ohshiro, Toshiyuki Matsubara, Masaru TanokuraAbstract: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.
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a novel enzyme 2 Hydroxybiphenyl 2 sulfinate desulfinase dszb from a dibenzothiophene desulfurizing bacterium rhodococcus erythropolis ka2 5 1 gene overexpression and enzyme characterization
Biochimica et Biophysica Acta, 2002Co-Authors: Norikazu Nakayama, Kenichi Koizumi, Masanori Suzuki, Takashi Ohshiro, Toshiyuki Matsubara, Yuko Moroto, Yasushi Kawata, Yasuto Hirakawa, Kenji Maruhashi, Yoshikazu IzumiAbstract: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.
Takashi Ohshiro - One of the best experts on this subject based on the ideXlab platform.
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crystal structures of apo dszc and fmn bound dszc from rhodococcus erythropolis d 1
FEBS Journal, 2015Co-Authors: Lijun Guan, Yoshikazu Izumi, Takashi Ohshiro, Woo Cheol Lee, Shipeng Wang, Jun Ohtsuka, Masaru TanokuraAbstract: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).
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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
Bioscience Biotechnology and Biochemistry, 2007Co-Authors: Takashi Ohshiro, Woo Cheol Lee, Masaru Tanokura, Ryo Ohkita, Takeshi Takikawa, Masanori Manabe, Yoshikazu IzumiAbstract: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.
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crystal structure and desulfurization mechanism of 2 Hydroxybiphenyl 2 sulfinic acid desulfinase
Journal of Biological Chemistry, 2006Co-Authors: Woo Cheol Lee, Yoshikazu Izumi, Takashi Ohshiro, Toshiyuki Matsubara, Masaru TanokuraAbstract: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 His60 to the active site. The sulfinate group of bound substrates forms hydrogen bonds with side chains of Ser27, His60, and Arg70, each of which is shown by site-directed mutagenesis to be essential for the activity. In our proposed reaction mechanism, Cys27 functions as a nucleophile and seems to be activated by the sulfinate group of substrates, whereas His60 and Arg70 orient the syn orbital of sulfinate oxygen to the sulfhydryl hydrogen of Cys27 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.
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crystallization and preliminary x ray analyses of desulfurization enzyme dszb and its c27s mutant complexed with biphenyl 2 sulfinic acid
Acta Crystallographica Section D-biological Crystallography, 2004Co-Authors: Woo Cheol Lee, Yoshikazu Izumi, Takashi Ohshiro, Toshiyuki Matsubara, Masaru TanokuraAbstract: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.
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a novel enzyme 2 Hydroxybiphenyl 2 sulfinate desulfinase dszb from a dibenzothiophene desulfurizing bacterium rhodococcus erythropolis ka2 5 1 gene overexpression and enzyme characterization
Biochimica et Biophysica Acta, 2002Co-Authors: Norikazu Nakayama, Kenichi Koizumi, Masanori Suzuki, Takashi Ohshiro, Toshiyuki Matsubara, Yuko Moroto, Yasushi Kawata, Yasuto Hirakawa, Kenji Maruhashi, Yoshikazu IzumiAbstract: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.
Masaru Tanokura - One of the best experts on this subject based on the ideXlab platform.
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crystal structures of apo dszc and fmn bound dszc from rhodococcus erythropolis d 1
FEBS Journal, 2015Co-Authors: Lijun Guan, Yoshikazu Izumi, Takashi Ohshiro, Woo Cheol Lee, Shipeng Wang, Jun Ohtsuka, Masaru TanokuraAbstract: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).
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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
Bioscience Biotechnology and Biochemistry, 2007Co-Authors: Takashi Ohshiro, Woo Cheol Lee, Masaru Tanokura, Ryo Ohkita, Takeshi Takikawa, Masanori Manabe, Yoshikazu IzumiAbstract: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.
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crystal structure and desulfurization mechanism of 2 Hydroxybiphenyl 2 sulfinic acid desulfinase
Journal of Biological Chemistry, 2006Co-Authors: Woo Cheol Lee, Yoshikazu Izumi, Takashi Ohshiro, Toshiyuki Matsubara, Masaru TanokuraAbstract: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 His60 to the active site. The sulfinate group of bound substrates forms hydrogen bonds with side chains of Ser27, His60, and Arg70, each of which is shown by site-directed mutagenesis to be essential for the activity. In our proposed reaction mechanism, Cys27 functions as a nucleophile and seems to be activated by the sulfinate group of substrates, whereas His60 and Arg70 orient the syn orbital of sulfinate oxygen to the sulfhydryl hydrogen of Cys27 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.
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crystallization and preliminary x ray analyses of desulfurization enzyme dszb and its c27s mutant complexed with biphenyl 2 sulfinic acid
Acta Crystallographica Section D-biological Crystallography, 2004Co-Authors: Woo Cheol Lee, Yoshikazu Izumi, Takashi Ohshiro, Toshiyuki Matsubara, Masaru TanokuraAbstract: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.
Christina M Payne - One of the best experts on this subject based on the ideXlab platform.
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inhibition mechanisms of rhodococcus erythropolis 2 Hydroxybiphenyl 2 sulfinate desulfinase dszb
Journal of Physical Chemistry B, 2019Co-Authors: Landon C Mills, Derek L Englert, Christina M PayneAbstract:Naturally occurring enzymatic pathways enable highly specific, rapid thiophenic sulfur cleavage occurring at ambient temperature and pressure, which may be harnessed for the desulfurization of petroleum-based fuel. One pathway found in bacteria is a four-step catabolic pathway (the 4S pathway) converting dibenzothiophene (DBT), a common crude oil contaminant, into 2-Hydroxybiphenyl (HBP) without disrupting the carbon-carbon bonds. 2'-Hydroxybiphenyl-2-sulfinate desulfinase (DszB), the rate-limiting enzyme in the enzyme cascade, is capable of selectively cleaving carbon-sulfur bonds. Accordingly, understanding the molecular mechanisms of DszB activity may enable development of the cascade as industrial biotechnology. Based on crystallographic evidence, we hypothesized that DszB undergoes an active site conformational change associated with the catalytic mechanism. Moreover, we anticipated this conformational change is responsible, in part, for enhancing product inhibition. Rhodococcus erythropolis IGTS8 DszB was recombinantly produced and purified via Escherichia coli BL21 to test these hypotheses. Activity and the resulting conformational change of DszB in the presence of HBP were evaluated. The activity of recombinant DszB was comparable to the natively expressed enzyme and was inhibited via competitive binding of the product, HBP. Using circular dichroism, global changes in DszB conformation were monitored in response to HBP concentration, which indicated that both product and substrate produced similar structural changes. Molecular dynamics (MD) simulations and free energy perturbation with Hamiltonian replica exchange molecular dynamics (FEP/λ-REMD) calculations were used to investigate the molecular-level phenomena underlying the connection between conformation change and kinetic inhibition. In addition to the HBP, MD simulations of DszB bound to common, yet structurally diverse, crude oil contaminants 2',2-biphenol (BIPH), 1,8-naphthosultam (NTAM), 2-biphenyl carboxylic acid (BCA), and 1,8-naphthosultone (NAPO) were performed. Analysis of the simulation trajectories, including root-mean-square fluctuation (RMSF), center of mass (COM) distances, and strength of nonbonded interactions, when compared with FEP/λ-REMD calculations of ligand binding free energy, showed excellent agreement with experimentally determined inhibition constants. Together, the results show that the combination of a molecule's hydrophobicity and nonspecific interactions with nearby functional groups contributes to a competitive inhibition mechanism that locks DszB in a closed conformation and precludes substrate access to the active site.
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desulfination by 2 Hydroxybiphenyl 2 sulfinate desulfinase proceeds via electrophilic aromatic substitution by the cysteine 27 proton
Chemical Science, 2017Co-Authors: Inacrist Geronimo, Shawn R Nigam, Christina M PayneAbstract:Biodesulfurization is an attractive option for enzymatically removing sulfur from the recalcitrant thiophenic derivatives that comprise the majority of organosulfur compounds remaining in hydrotreated petroleum products. Desulfurization in the bacteria Rhodococcus erythropolis follows a four-step pathway culminating in C-S bond cleavage in the 2'-Hydroxybiphenyl-2-sulfinate (HBPS) intermediate to yield 2-Hydroxybiphenyl and bisulfite. The reaction, catalyzed by 2'-Hydroxybiphenyl-2-sulfinate desulfinase (DszB), is the rate-limiting step and also the least understood, as experimental evidence points to a mechanism unlike that of other desulfinases. On the basis of structural and biochemical evidence, two possible mechanisms have been proposed: nucleophilic addition and electrophilic aromatic substitution. Density functional theory calculations showed that electrophilic substitution by a proton is the lower energy pathway and is consistent with previous kinetic and site-directed mutagenesis studies. C27 transfers its proton to HBPS, leading directly to the release of SO2 without the formation of a carbocation intermediate. The H60-S25 dyad stabilizes the transition state by withdrawing the developing negative charge on cysteine. Establishing the desulfination mechanism and specific role of active site residues, accomplished in this study, is essential to protein engineering efforts to increase DszB catalytic activity, which is currently too low for industrial-scale application.
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charmm force field parameters for 2 Hydroxybiphenyl 2 sulfinate 2 Hydroxybiphenyl and related analogs
Journal of Molecular Graphics & Modelling, 2017Co-Authors: Ishan Fursule, Landon C Mills, Derek L Englert, Brad J Berron, Christina M PayneAbstract:Abstract 2′-Hydroxybiphenyl-2-sulfinate (HBPS) desulfinase (DszB) catalyzes the cleavage of the carbon-sulfur bond from HBPS in the final step of microbial 4S pathway desulfurization reactions. DszB is notable for its substrate specificity and exhibits product inhibition, both of which hinder the overall 4S pathway turnover rate. To understand the molecular-level contributions to substrate and inhibitor binding to DszB, we plan to perform molecular dynamic simulations bound to an array of naphthenic molecules and biphenyl analogues of HBPS. However, many of the small molecules we are interested in are not included in standard force field packages, and thus, we must first produce accurate molecular mechanics force fields. Here, we develop and validate CHARMM-compatible force field parameters for the HBPS substrate, the 2-Hydroxybiphenyl product, and potential inhibitors including: 2,2′-biphenol, 2-biphenyl carboxylic acid, 1,8-naphthosultam, and 1,8-naphthosultone. The selected molecules represent biphenyl compounds having both a single and double functional group and the planar naphthenic molecule class, all likely present in the oil-rich environment surrounding DszB-producing microorganisms. The Force Field Toolkit (ffTK) in VMD was used to optimize charge, bond distance, angle, and dihedral parameters. Optimized geometries were determined from quantum mechanical calculations. Molecular simulations of the molecules in explicit and implicit water solutions were conducted to assess the abilities of optimized parameters to recapitulate optimized geometries. Calculated infrared (IR) spectra were obtained and compared with experimental IR spectra for validation of the optimized MM parameters.