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Yuji Nagata - One of the best experts on this subject based on the ideXlab platform.
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degradation of β hexachlorocyclohexane by haloalkane dehalogenase linb from Sphingomonas paucimobilis ut26
Applied and Environmental Microbiology, 2005Co-Authors: Yuji Nagata, Yoshiyuki Ohtsubo, Petr Jerabek, Yukari Sato, Ashwani Kumar, Masataka TsudaAbstract:β-Hexachlorocyclohexane (β-HCH) is the most recalcitrant among the α-, β-, γ-, and δ-isomers of HCH and causes serious environmental pollution problems. We demonstrate here that the haloalkane dehalogenase LinB, reported earlier to mediate the second step in the degradation of γ-HCH in Sphingomonas paucimobilis UT26, metabolizes β-HCH to produce 2,3,4,5,6-pentachlorocyclohexanol.
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quantitative analysis of substrate specificity of haloalkane dehalogenase linb from Sphingomonas paucimobilis ut26
Biochemistry, 2005Co-Authors: Jan Kmuníček, Kamila Hynkova, Yuji Nagata, Tomas Jedlička, Ana Negri, Federico Gago, Rebecca C. WadeAbstract:Haloalkane dehalogenases are microbial enzymes that cleave a carbon-halogen bond in halogenated compounds. The haloalkane dehalogenase LinB, isolated from Sphingomonas paucimobilis UT26, is a broad-specificity enzyme. Fifty-five halogenated aliphatic and cyclic hydrocarbons were tested for dehalogenation with the LinB enzyme. The compounds for testing were systematically selected using a statistical experimental design. Steady-state kinetic constants Km and kcat were determined for 25 substrates that showed detectable cleavage by the enzyme and low abiotic hydrolysis. Classical quantitative structure - activity relationships (QSARs) were used to correlate the kinetic constants with molecular descriptors and resulted in a model that explained 94% of the experimental data variability. The binding affinity of the tested substrates for this haloalkane dehalogenase correlated with hydrophobicity, molecular surface, dipole moment, and volume:surface ratio. Binding of the substrate molecules in the active site pocket of LinB depends nonlinearly on the size of the molecules. Binding affinity increases with increasing substrate size up to a chain length of six carbon atoms and then decreases. Comparative binding energy (COMBINE) analysis was then used to identify amino acid residues in LinB that modulate its substrate specificity. A model with three statistically significant principal components explained 95% of the experimental data variability. van der Waals interactions between substrate molecules and the enzyme dominated the COMBINE model, in agreement with the importance of substrate size in the classical QSAR model. Only a limited number of protein residues (6-8%) contribute significantly to the explanation of variability in binding affinities. The amino acid residues important for explaining variability in binding affinities are as follows: (i) first-shell residues Asn38, Asp108, Trp109, Glu132, Ile134, Phe143, Phe151, Phe169, Val173, Trp207, Pro208, Ile211, Leu248, and His272, (ii) tunnel residues Pro144, Asp147, Leu177, and Ala247, and (iii) second-shell residues Pro39 and Phe273. The tunnel and the second-shell residues represent the best targets for modulating specificity since their replacement does not lead to loss of functionality by disruption of the active site architecture. The mechanism of molecular adaptation toward a different specificity is discussed on the basis of quantitative comparison of models derived for two protein family members.
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identification and characterization of genes involved in the downstream degradation pathway of γ hexachlorocyclohexane in Sphingomonas paucimobilis ut26
Journal of Bacteriology, 2005Co-Authors: Ryo Endo, Yoshiyuki Ohtsubo, Masataka Tsuda, Keisuke Miyauchi, Masao Fukuda, Mayuko Kamakura, Yuji NagataAbstract:Sphingomonas paucimobilis UT26 utilizes γ-hexachlorocyclohexane (γ-HCH) as a sole source of carbon and energy. In our previous study, we cloned and characterized genes that are involved in the conversion of γ-HCH to maleylacetate (MA) via chlorohydroquinone (CHQ) in UT26. In this study, we identified and characterized an MA reductase gene, designated linF, that is essential for the utilization of γ-HCH in UT26. A gene named linEb, whose deduced product showed significant identity to LinE (53%), was located close to linF. LinE is a novel type of ring cleavage dioxygenase that catalyzes the conversion of CHQ to MA. LinEb expressed in Escherichia coli transformed CHQ and 2,6-dichlorohydroquinone to MA and 2-chloromaleylacetate, respectively. Our previous and present results indicate that UT26 (i) has two gene clusters for degradation of chlorinated aromatic compounds via hydroquinone-type intermediates and (ii) uses at least parts of both clusters for γ-HCH utilization.
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crystal structure of haloalkane dehalogenase linb from Sphingomonas paucimobilis ut26 at 0 95 a resolution dynamics of catalytic residues
Biochemistry, 2004Co-Authors: Aaron J Oakley, Yuji Nagata, Martin Klvaňa, Matthew Charles James Wilce, Michal Otyepka, Jiri DamborskyAbstract:We present the structure of LinB, a 33-kDa haloalkane dehalogenase from Sphingomonas paucimobilis UT26, at 0.95 A resolution. The data have allowed us to directly observe the anisotropic motions of the catalytic residues. In particular, the side-chain of the catalytic nucleophile, Asp108, displays a high degree of disorder. It has been modeled in two conformations, one similar to that observed previously (conformation A) and one strained (conformation B) that approached the catalytic base (His272). The strain in conformation B was mainly in the CR-C‚-CA angle (126°) that deviated by 13.4° from the "ideal" bond angle of 112.6 °. On the basis of these observations, we propose a role for the charge state of the catalytic histidine in determining the geometry of the catalytic residues. We hypothesized that double-protonation of the catalytic base (His272) reduces the distance between the side-chain of this residue and that of the Asp108. The results of molecular dynamics simulations were consistent with the structural data showing that protonation of the His272 side-chain nitrogen atoms does indeed reduce the distance between the side-chains of the residues in question, although the simulations failed to demonstrate the same degree of strain in the Asp108 CR-C‚-CA angle. Instead, the changes in the molecular dynamics structures were distributed over several bond and dihedral angles. Quantum mechanics calculations on LinB with 1-chloro-2,2-dimethylpropane as a substrate were performed to determine which active site conformations and protonation states were most likely to result in catalysis. It was shown that His272 singly protonated at N‰1 and Asp108 in conformation A gave the most exothermic reaction (¢H )- 22 kcal/mol). With His272 doubly protonated at N‰1 and N2, the reactions were only slightly exothermic or were endothermic. In all calculations starting with Asp108 in conformation B, the Asp108 CR-C‚-CA angle changed during the reaction and the Asp108 moved to conformation A. The results presented here indicate that the positions of the catalytic residues and charge state of the catalytic base are important for determining reaction energetics in LinB.
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modification of activity and specificity of haloalkane dehalogenase from Sphingomonas paucimobilis ut26 by engineering of its entrance tunnel
Journal of Biological Chemistry, 2003Co-Authors: Radka Chaloupkova, Jana Sýkorova, Marta Monincova, Martina Pavlová, Andrea Jesenska, Masataka Tsuda, Zbynek Prokop, Yuji NagataAbstract:Abstract Structural comparison of three different haloalkane dehalogenases suggested that substrate specificity of these bacterial enzymes could be significantly influenced by the size and shape of their entrance tunnels. The surface residue leucine 177 positioned at the tunnel opening of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 was selected for modification based on structural and phylogenetic analysis; the residue partially blocks the entrance tunnel, and it is the most variable pocket residue in haloalkane dehalogenase-like proteins with nine substitutions in 14 proteins. Mutant genes coding for proteins carrying all possible substitutions in position 177 were constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli. In total, 15 active protein variants were obtained, suggesting a relatively high tolerance of the site for the introduction of mutations. Purified protein variants were kinetically characterized by determination of specific activities with 12 halogenated substrates and steady-state kinetic parameters with two substrates. The effect of mutation on the enzyme activities varied dramatically with the structure of the substrates, suggesting that extrapolation of one substrate to another may be misleading and that a systematic characterization of the protein variants with a number of substrates is essential. Multivariate analysis of activity data revealed that catalytic activity of mutant enzymes generally increased with the introduction of small and nonpolar amino acid in position 177. This result is consistent with the phylogenetic analysis showing that glycine and alanine are the most commonly occurring amino acids in this position among haloalkane dehalogenases. The study demonstrates the advantages of using rational engineering to develop enzymes with modified catalytic properties and substrate specificities. The strategy of using site-directed mutagenesis to modify a specific entrance tunnel residue identified by structural and phylogenetic analyses, rather than combinatorial screening, generated a high percentage of viable mutants.
Masahiro Takagi - One of the best experts on this subject based on the ideXlab platform.
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crystallization and preliminary x ray diffraction analysis of haloalkane dehalogenase linb from Sphingomonas paucimobilis ut26
Acta Crystallographica Section D-biological Crystallography, 1999Co-Authors: Ivana Kuta Smatanova, Yuji Nagata, Masahiro Takagi, L A Svensson, Jaromír MarekAbstract:Haloalkane hydrolytic dehalogenase LinB from Sphingomonas paucimobilis UT26, an enzyme which releases chloride or bromide anion from n-halogenated alkanes and has a broad range of substrate specificity, was crystallized using the hanging-drop vapour-diffusion method at 278 K. The best crystals were obtained by microseeding with a precipitant containing 18–20%(w/v) PEG 6000, 0.2 M calcium acetate and 0.1 M Tris–HCl pH 8.9. The crystals diffract to at least 1.60 A using synchrotron X-ray under cryogenic (100 K) conditions. They belong to the orthorhombic space group P21212 with unit-cell parameters a = 50.29, b = 71.70, c = 72.73 A. The asymmetric unit contains one molecule of the enzyme.
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purification and characterization of a haloalkane dehalogenase of a new substrate class from a gamma hexachlorocyclohexane degrading bacterium Sphingomonas paucimobilis ut26
Applied and Environmental Microbiology, 1997Co-Authors: Yuji Nagata, Katka Manova, Alena Ansorgová, Keisuke Miyauchi, Masahiro TakagiAbstract:The linB gene product (LinB), 1,3,4,6-tetrachloro-1,4-cyclohexadiene halidohydrolase, which is involved in the degradation of gamma-hexachlorocyclohexane in Sphingomonas paucimobilis UT26 (Y. Nagata, T. Nariya, R. Ohtomo, M. Fukuda, K. Yano, and M. Takagi, J. Bacteriol. 175:6403-6410, 1993), was overproduced in E. coli and purified to homogeneity. The molecular mass of LinB was deduced to be 30 kDa by gel filtration chromatography and 32 kDa by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel, indicating that LiuB is a monomeric enzyme. The optimal pH for activity was 8.2. Not only monochloroalkanes (C3 to C10) but also dichloroalkanes, bromoalkanes, and chlorinated allphatic alcohols were good substrates for LinB, suggesting that LinB shares properties with another haloalkane dehalogenase, DhlA (S. Keuning, D.B. Janssen, and B. Witholt, J. Bacteriol. 163:635-639, 1985), which shows significant similarity to LinB in primary structure (D. B. Janssen, F. Pries, J. van der Ploeg, B. Kazemier, P. Terpstra, and B. Witholt, J. Bacteriol. 171:6791-6799, 1989) but not in substrate specificity. Principal component analysis of substrate activities of various haloalkane dehalogenases suggested that LinB probably constitutes a new substrate specificity class within this group of enzymes.
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purification and characterization of a haloalkane dehalogenase of a new substrate class from a gamma hexachlorocyclohexane degrading bacterium Sphingomonas paucimobilis ut26
Applied and Environmental Microbiology, 1997Co-Authors: Yuji Nagata, Katka Manova, Alena Ansorgová, Keisuke Miyauchi, Jiri Damborsky, Masahiro TakagiAbstract:The linB gene product (LinB), 1,3,4,6-tetrachloro-1,4-cyclohexadiene halidohydrolase, which is involved in the degradation of gamma-hexachlorocyclohexane in Sphingomonas paucimobilis UT26 (Y. Nagata, T. Nariya, R. Ohtomo, M. Fukuda, K. Yano, and M. Takagi, J. Bacteriol. 175:6403-6410, 1993), was overproduced in E. coli and purified to homogeneity. The molecular mass of LinB was deduced to be 30 kDa by gel filtration chromatography and 32 kDa by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel, indicating that LiuB is a monomeric enzyme. The optimal pH for activity was 8.2. Not only monochloroalkanes (C3 to C10) but also dichloroalkanes, bromoalkanes, and chlorinated allphatic alcohols were good substrates for LinB, suggesting that LinB shares properties with another haloalkane dehalogenase, DhlA (S. Keuning, D.B. Janssen, and B. Witholt, J. Bacteriol. 163:635-639, 1985), which shows significant similarity to LinB in primary structure (D. B. Janssen, F. Pries, J. van der Ploeg, B. Kazemier, P. Terpstra, and B. Witholt, J. Bacteriol. 171:6791-6799, 1989) but not in substrate specificity. Principal component analysis of substrate activities of various haloalkane dehalogenases suggested that LinB probably constitutes a new substrate specificity class within this group of enzymes.
Keisuke Miyauchi - One of the best experts on this subject based on the ideXlab platform.
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a tetrahydrofolate dependent o demethylase ligm is crucial for catabolism of vanillate and syringate in Sphingomonas paucimobilis syk 6
Journal of Bacteriology, 2005Co-Authors: Eiji Masai, Keisuke Miyauchi, Yoshihiro Katayama, Masao FukudaAbstract:Vanillate and syringate are converted into protocatechuate (PCA) and 3-O-methylgallate (3MGA), respectively, by O-demethylases in Sphingomonas paucimobilis SYK-6. PCA is further degraded via the PCA 4,5-cleavage pathway, while 3MGA is degraded through multiple pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and an unidentified 3MGA O-demethylase and gallate dioxygenase are participants. For this study, we isolated a 4.7-kb SmaI fragment that conferred on Escherichia coli the activity required for the conversion of vanillate to PCA. The nucleotide sequence of this fragment revealed an open reading frame of 1,413 bp (ligM), the deduced amino acid sequence of which showed 49% identity with that of the tetrahydrofolate (H4folate)-dependent syringate O-demethylase gene (desA). The metF and ligH genes, which are thought to be involved in H4folate-mediated C1 metabolism, were located just downstream of ligM. The crude LigM enzyme expressed in E. coli converted vanillate and 3MGA to PCA and gallate, respectively, with similar specific activities, and only in the presence of H4folate; however, syringate was not a substrate for LigM. The disruption of ligM led to significant growth retardation on both vanillate and syringate, indicating that ligM is involved in the catabolism of these substrates. The ability of the ligM mutant to transform vanillate was markedly decreased, and this mutant completely lost the 3MGA O-demethylase activity. A ligM desA double mutant completely lost the ability to transform vanillate, thus indicating that desA also contributes to vanillate degradation. All of these results indicate that ligM encodes vanillate/3MGA O-demethylase and plays an important role in the O demethylation of vanillate and 3MGA, respectively.
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identification and characterization of genes involved in the downstream degradation pathway of γ hexachlorocyclohexane in Sphingomonas paucimobilis ut26
Journal of Bacteriology, 2005Co-Authors: Ryo Endo, Yoshiyuki Ohtsubo, Masataka Tsuda, Keisuke Miyauchi, Masao Fukuda, Mayuko Kamakura, Yuji NagataAbstract:Sphingomonas paucimobilis UT26 utilizes γ-hexachlorocyclohexane (γ-HCH) as a sole source of carbon and energy. In our previous study, we cloned and characterized genes that are involved in the conversion of γ-HCH to maleylacetate (MA) via chlorohydroquinone (CHQ) in UT26. In this study, we identified and characterized an MA reductase gene, designated linF, that is essential for the utilization of γ-HCH in UT26. A gene named linEb, whose deduced product showed significant identity to LinE (53%), was located close to linF. LinE is a novel type of ring cleavage dioxygenase that catalyzes the conversion of CHQ to MA. LinEb expressed in Escherichia coli transformed CHQ and 2,6-dichlorohydroquinone to MA and 2-chloromaleylacetate, respectively. Our previous and present results indicate that UT26 (i) has two gene clusters for degradation of chlorinated aromatic compounds via hydroquinone-type intermediates and (ii) uses at least parts of both clusters for γ-HCH utilization.
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a novel tetrahydrofolate dependent o demethylase gene is essential for growth of Sphingomonas paucimobilis syk 6 with syringate
Journal of Bacteriology, 2004Co-Authors: Eiji Masai, Keisuke Miyauchi, Tomonori Sonoki, Yoshihiro Katayama, Miyuki Sasaki, Yasunori Minakawa, Masao FukudaAbstract:Sphingomonas paucimobilis SYK-6 degrades syringate to 3-O-methylgallate (3MGA), which is finally converted to pyruvate and oxaloacetate via multiple pathways in which protocatechuate 4,5-dioxygenase, 3MGA dioxygenase, and gallate dioxygenase are involved. Here we isolated the syringate O-demethylase gene (desA), which complemented the growth deficiency on syringate of a Tn5 mutant of the SYK-6 derivative strain. The desA gene is located 929 bp downstream of ferA, encoding feruloyl-coenzyme A synthetase, and consists of a 1,386-bp open reading frame encoding a polypeptide with a molecular mass of 50,721 Da. The deduced amino acid sequence of desA showed 26% identity in a 325-amino-acid overlap with that of gcvT of Escherichia coli, which encodes the tetrahydrofolate (H4folate)-dependent aminomethyltransferase involved in glycine cleavage. The cell extract of E. coli carrying desA converted syringate to 3MGA only when H4folate was added to the reaction mixture. DesA catalyzes the transfer of the methyl moiety of syringate to H4folate, forming 5-methyl-H4folate. Vanillate and 3MGA were also used as substrates for DesA; however, the relative activities toward them were 3 and 0.4% of that toward syringate, respectively. Disruption of desA in SYK-6 resulted in a growth defect on syringate but did not affect growth on vanillate, indicating that desA is essential to syringate degradation. In a previous study the ligH gene, which complements the growth deficiency on vanillate and syringate of a chemical-induced mutant of SYK-6, DC-49, was isolated (S. Nishikawa, T. Sonoki, T. Kasahara, T. Obi, S. Kubota, S. Kawai, N. Morohoshi, and Y. Katayama, Appl. Environ. Microbiol. 64:836-842, 1998). Disruption of ligH resulted in the same phenotype as DC-49; its cell extract, however, was found to be able to convert vanillate and syringate in the presence of H4folate. The possible role of ligH is discussed.
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purification and characterization of a haloalkane dehalogenase of a new substrate class from a gamma hexachlorocyclohexane degrading bacterium Sphingomonas paucimobilis ut26
Applied and Environmental Microbiology, 1997Co-Authors: Yuji Nagata, Katka Manova, Alena Ansorgová, Keisuke Miyauchi, Masahiro TakagiAbstract:The linB gene product (LinB), 1,3,4,6-tetrachloro-1,4-cyclohexadiene halidohydrolase, which is involved in the degradation of gamma-hexachlorocyclohexane in Sphingomonas paucimobilis UT26 (Y. Nagata, T. Nariya, R. Ohtomo, M. Fukuda, K. Yano, and M. Takagi, J. Bacteriol. 175:6403-6410, 1993), was overproduced in E. coli and purified to homogeneity. The molecular mass of LinB was deduced to be 30 kDa by gel filtration chromatography and 32 kDa by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel, indicating that LiuB is a monomeric enzyme. The optimal pH for activity was 8.2. Not only monochloroalkanes (C3 to C10) but also dichloroalkanes, bromoalkanes, and chlorinated allphatic alcohols were good substrates for LinB, suggesting that LinB shares properties with another haloalkane dehalogenase, DhlA (S. Keuning, D.B. Janssen, and B. Witholt, J. Bacteriol. 163:635-639, 1985), which shows significant similarity to LinB in primary structure (D. B. Janssen, F. Pries, J. van der Ploeg, B. Kazemier, P. Terpstra, and B. Witholt, J. Bacteriol. 171:6791-6799, 1989) but not in substrate specificity. Principal component analysis of substrate activities of various haloalkane dehalogenases suggested that LinB probably constitutes a new substrate specificity class within this group of enzymes.
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purification and characterization of a haloalkane dehalogenase of a new substrate class from a gamma hexachlorocyclohexane degrading bacterium Sphingomonas paucimobilis ut26
Applied and Environmental Microbiology, 1997Co-Authors: Yuji Nagata, Katka Manova, Alena Ansorgová, Keisuke Miyauchi, Jiri Damborsky, Masahiro TakagiAbstract:The linB gene product (LinB), 1,3,4,6-tetrachloro-1,4-cyclohexadiene halidohydrolase, which is involved in the degradation of gamma-hexachlorocyclohexane in Sphingomonas paucimobilis UT26 (Y. Nagata, T. Nariya, R. Ohtomo, M. Fukuda, K. Yano, and M. Takagi, J. Bacteriol. 175:6403-6410, 1993), was overproduced in E. coli and purified to homogeneity. The molecular mass of LinB was deduced to be 30 kDa by gel filtration chromatography and 32 kDa by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel, indicating that LiuB is a monomeric enzyme. The optimal pH for activity was 8.2. Not only monochloroalkanes (C3 to C10) but also dichloroalkanes, bromoalkanes, and chlorinated allphatic alcohols were good substrates for LinB, suggesting that LinB shares properties with another haloalkane dehalogenase, DhlA (S. Keuning, D.B. Janssen, and B. Witholt, J. Bacteriol. 163:635-639, 1985), which shows significant similarity to LinB in primary structure (D. B. Janssen, F. Pries, J. van der Ploeg, B. Kazemier, P. Terpstra, and B. Witholt, J. Bacteriol. 171:6791-6799, 1989) but not in substrate specificity. Principal component analysis of substrate activities of various haloalkane dehalogenases suggested that LinB probably constitutes a new substrate specificity class within this group of enzymes.
Christof Holliger - One of the best experts on this subject based on the ideXlab platform.
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enantioselective transformation of α hexachlorocyclohexane by the dehydrochlorinases lina1 and lina2 from the soil bacterium Sphingomonas paucimobilis b90a
Applied and Environmental Microbiology, 2005Co-Authors: Mrutyunjay Suar, Andrea Hauser, Thomas Poiger, Hansrudolf Buser, Markus D Muller, Charu Dogra, Vishakha Raina, Christof Holliger, Jan Roelof Van Der MeerAbstract:Sphingomonas paucimobilis B90A contains two variants, LinA1 and LinA2, of a dehydrochlorinase that catalyzes the first and second steps in the metabolism of hexachlorocyclohexanes (R. Kumari, S. Subudhi, M. Suar, G. Dhingra, V. Raina, C. Dogra, S. Lal, J. R. van der Meer, C. Holliger, and R. Lal, Appl. Environ. Microbiol. 68:6021-6028, 2002). On the amino acid level, LinA1 and LinA2 were 88% identical to each other, and LinA2 was 100% identical to LinA of S. paucimobilis UT26. Incubation of chiral α-hexachlorocyclohexane (α-HCH) with Escherichia coli BL21 expressing functional LinA1 and LinA2 S-glutathione transferase fusion proteins showed that LinA1 preferentially converted the (+) enantiomer, whereas LinA2 preferred the (−) enantiomer. Concurrent formation and subsequent dissipation of β-pentachlorocyclohexene enantiomers was also observed in these experiments, indicating that there was enantioselective formation and/or dissipation of these enantiomers. LinA1 preferentially formed (3S,4S,5R,6R)-1,3,4,5,6-pentachlorocyclohexene, and LinA2 preferentially formed (3R,4R,5S,6S)-1,3,4,5,6-pentachlorocyclohexene. Because enantioselectivity was not observed in incubations with whole cells of S. paucimobilis B90A, we concluded that LinA1 and LinA2 are equally active in this organism. The enantioselective transformation of chiral α-HCH by LinA1 and LinA2 provides the first evidence of the molecular basis for the changed enantiomer composition of α-HCH in many natural environments. Enantioselective degradation may be one of the key processes determining enantiomer composition, especially when strains that contain only one of the linA genes, such as S. paucimobilis UT26, prevail.
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hexachlorocyclohexane degrading bacterial strains Sphingomonas paucimobilis b90a ut26 and sp having similar lin genes represent three distinct species sphingobium indicum sp nov sphingobium japonicum sp nov and sphingobium francense sp nov and reclas
International Journal of Systematic and Evolutionary Microbiology, 2005Co-Authors: Rinku Pal, Shashi Bala, Mandeep Dadhwal, Mukesh Kumar, Gauri Dhingra, Om Prakash, Solai Ramatchandirane Prabagaran, S Shivaji, John Cullum, Christof HolligerAbstract:Three strains of Sphingomonas paucimobilis, B90A, UT26 and Sp+, isolated from different geographical locations, were found to degrade hexachlorocyclohexane. Phylogenetic analysis based on 16S rRNA gene sequences indicated that these strains do not fall in a clade that includes the type strain, Sphingomonas paucimobilis ATCC 29837T, but form a coherent cluster with [Sphingomonas] chungbukensis IMSNU 11152T followed by Sphingobium chlorophenolicum ATCC 33790T. The three strains showed low DNA–DNA relatedness values with Sphingomonas paucimobilis ATCC 29837T (8–25 %), [Sphingomonas] chungbukensis IMSNU 11152T (10–17 %), Sphingobium chlorophenolicum ATCC 33790T (23–54 %) and Sphingomonas xenophaga DSM 6383T (10–28 %), indicating that they do not belong to any of these species. Although the three strains were found to be closely related to each other based on 16S rRNA gene sequence similarity (99·1–99·4 %), DNA–DNA relatedness (19–59 %) and pulsed-field gel electrophoresis (PFGE) patterns indicated that they possibly represent three novel species of the genus Sphingobium. The three strains could also be readily distinguished by biochemical tests. The three strains showed similar polar lipid profiles and contained sphingoglycolipids. The strains differed from each other in fatty acid composition but contained the predominant fatty acids characteristic of other Sphingobium species. A phylogenetic study based on 16S rRNA gene sequences showed that [Sphingomonas] chungbukensis IMSNU 11152T formed a cluster with members of the genus Sphingobium. Based on these results, it is proposed that strains B90A, UT26 and Sp+, previously known as Sphingomonas paucimobilis, are the type strains of Sphingobium indicum sp. nov. (=MTCC 6364T=CCM 7286T), Sphingobium japonicum sp. nov. (=MTCC 6362T=CCM 7287T) and Sphingobium francense sp. nov. (=MTCC 6363T=CCM 7288T), respectively. It is also proposed that [Sphingomonas] chungbukensis be transferred to Sphingobium chungbukense comb. nov.
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organization of lin genes and is6100 among different strains of hexachlorocyclohexane degrading Sphingomonas paucimobilis evidence for horizontal gene transfer
Journal of Bacteriology, 2004Co-Authors: Charu Dogra, Mrutyunjay Suar, Vishakha Raina, Christof Holliger, Jan Roelof Van Der Meer, Rinku Pal, Sukanya Lal, Karlheinz Gartemann, Rup LalAbstract:The organization of lin genes and IS6100 was studied in three strains of Sphingomonas paucimobilis (B90A, Sp+, and UT26) which degraded hexachlorocyclohexane (HCH) isomers but which had been isolated at different geographical locations. DNA-DNA hybridization data revealed that most of the lin genes in these strains were associated with IS6100, an insertion sequence classified in the IS6 family and initially found in Mycobacterium fortuitum. Eleven, six, and five copies of IS6100 were detected in B90A, Sp+, and UT26, respectively. IS6100 elements in B90A were sequenced from five, one, and one regions of the genomes of B90A, Sp+, and UT26, respectively, and were found to be identical. DNA-DNA hybridization and DNA sequencing of cosmid clones also revealed that S. paucimobilis B90A contains three and two copies of linX and linA, respectively, compared to only one copy of these genes in strains Sp+ and UT26. Although the copy number and the sequence of the remaining genes of the HCH degradative pathway (linB, linC, linD, and linE) were nearly the same in all strains, there were striking differences in the organization of the linA genes as a result of replacement of portions of DNA sequences by IS6100, which gave them a strange mosaic configuration. Spontaneous deletion of linD and linE from B90A and of linA from Sp+ occurred and was associated either with deletion of a copy of IS6100 or changes in IS6100 profiles. The evidence gathered in this study, coupled with the observation that the G+C contents of the linA genes are lower than that of the remaining DNA sequence of S. paucimobilis, strongly suggests that all these strains acquired the linA gene through horizontal gene transfer mediated by IS6100. The association of IS6100 with the rest of the lin genes further suggests that IS6100 played a role in shaping the current lin gene organization.
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cloning and characterization of lin genes responsible for the degradation of hexachlorocyclohexane isomers by Sphingomonas paucimobilis strain b90
Applied and Environmental Microbiology, 2002Co-Authors: Rekha Kumari, Mrutyunjay Suar, Charu Dogra, Vishakha Raina, Christof Holliger, Jan Roelof Van Der Meer, Gauri Dhingra, Sanjukta Subudhi, Sukanya Lal, Rup LalAbstract:Hexachlorocyclohexane (HCH) has been used extensively against agricultural pests and in public health programs for the control of mosquitoes. Commercial formulations of HCH consist of a mixture of four isomers, alpha, beta, gamma, and delta. While all these isomers pose serious environmental problems, beta-HCH is more problematic due to its longer persistence in the environment. We have studied the degradation of HCH isomers by Sphingomonas paucimobilis strain B90 and characterized the lin genes encoding enzymes from strain B90 responsible for the degradation of HCH isomers. Two nonidentical copies of the linA gene encoding HCH dehydrochlorinase, which were designated linA1 and linA2, were found in S. paucimobilis B90. The linA1 and linA2 genes could be expressed in Escherichia coli, leading to dehydrochlorination of alpha-, gamma-, and delta-HCH but not of beta-HCH, suggesting that S. paucimobilis B90 contains another pathway for the initial steps of beta-HCH degradation. The cloning and characterization of the halidohydrolase (linB), dehydrogenase (linC and linX), and reductive dechlorinase (linD) genes from S. paucimobilis B90 revealed that they share approximately 96 to 99% identical nucleotides with the corresponding genes of S. paucimobilis UT26. No evidence was found for the presence of a linE-like gene, coding for a ring cleavage dioxygenase, in strain B90. The gene structures around the linA1 and linA2 genes of strain B90, compared to those in strain UT26, are suggestive of a recombination between linA1 and linA2, which formed linA of strain UT26.
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degradation of 3 o methylgallate in Sphingomonas paucimobilis syk 6 by pathways involving protocatechuate 4 5 dioxygenase
Fems Microbiology Letters, 2007Co-Authors: Daisuke Kasai, Eiji Masai, Yoshihiro Katayama, Masao FukudaAbstract:Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate and 3-O-methylgallate (3MGA), respectively. 3MGA is metabolized via multiple pathways involving 3MGA 3,4-dioxygenase, protocatechuate 4,5-dioxygenase (LigAB), and gallate dioxygenase whereas protocatechuate is degraded via the protocatechuate 4,5-cleavage pathway. Here the secondary role of LigAB in syringate metabolism is investigated. The reaction product of 3MGA catalyzed by His-tagged LigAB was identified as 4-carboxy-2-hydroxy-6-methoxy-6-oxohexa-2,4-dienoate (CHMOD) and 2-pyrone-4,6-dicarboxylate (PDC), indicating that 3MGA is transformed to CHMOD and PDC by both reactions catalyzed by DesZ and LigAB. Mutant analysis revealed that the 3MGA catabolic pathways involving LigAB are functional in SYK-6.
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a tetrahydrofolate dependent o demethylase ligm is crucial for catabolism of vanillate and syringate in Sphingomonas paucimobilis syk 6
Journal of Bacteriology, 2005Co-Authors: Eiji Masai, Keisuke Miyauchi, Yoshihiro Katayama, Masao FukudaAbstract:Vanillate and syringate are converted into protocatechuate (PCA) and 3-O-methylgallate (3MGA), respectively, by O-demethylases in Sphingomonas paucimobilis SYK-6. PCA is further degraded via the PCA 4,5-cleavage pathway, while 3MGA is degraded through multiple pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and an unidentified 3MGA O-demethylase and gallate dioxygenase are participants. For this study, we isolated a 4.7-kb SmaI fragment that conferred on Escherichia coli the activity required for the conversion of vanillate to PCA. The nucleotide sequence of this fragment revealed an open reading frame of 1,413 bp (ligM), the deduced amino acid sequence of which showed 49% identity with that of the tetrahydrofolate (H4folate)-dependent syringate O-demethylase gene (desA). The metF and ligH genes, which are thought to be involved in H4folate-mediated C1 metabolism, were located just downstream of ligM. The crude LigM enzyme expressed in E. coli converted vanillate and 3MGA to PCA and gallate, respectively, with similar specific activities, and only in the presence of H4folate; however, syringate was not a substrate for LigM. The disruption of ligM led to significant growth retardation on both vanillate and syringate, indicating that ligM is involved in the catabolism of these substrates. The ability of the ligM mutant to transform vanillate was markedly decreased, and this mutant completely lost the 3MGA O-demethylase activity. A ligM desA double mutant completely lost the ability to transform vanillate, thus indicating that desA also contributes to vanillate degradation. All of these results indicate that ligM encodes vanillate/3MGA O-demethylase and plays an important role in the O demethylation of vanillate and 3MGA, respectively.
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identification and characterization of genes involved in the downstream degradation pathway of γ hexachlorocyclohexane in Sphingomonas paucimobilis ut26
Journal of Bacteriology, 2005Co-Authors: Ryo Endo, Yoshiyuki Ohtsubo, Masataka Tsuda, Keisuke Miyauchi, Masao Fukuda, Mayuko Kamakura, Yuji NagataAbstract:Sphingomonas paucimobilis UT26 utilizes γ-hexachlorocyclohexane (γ-HCH) as a sole source of carbon and energy. In our previous study, we cloned and characterized genes that are involved in the conversion of γ-HCH to maleylacetate (MA) via chlorohydroquinone (CHQ) in UT26. In this study, we identified and characterized an MA reductase gene, designated linF, that is essential for the utilization of γ-HCH in UT26. A gene named linEb, whose deduced product showed significant identity to LinE (53%), was located close to linF. LinE is a novel type of ring cleavage dioxygenase that catalyzes the conversion of CHQ to MA. LinEb expressed in Escherichia coli transformed CHQ and 2,6-dichlorohydroquinone to MA and 2-chloromaleylacetate, respectively. Our previous and present results indicate that UT26 (i) has two gene clusters for degradation of chlorinated aromatic compounds via hydroquinone-type intermediates and (ii) uses at least parts of both clusters for γ-HCH utilization.
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a novel tetrahydrofolate dependent o demethylase gene is essential for growth of Sphingomonas paucimobilis syk 6 with syringate
Journal of Bacteriology, 2004Co-Authors: Eiji Masai, Keisuke Miyauchi, Tomonori Sonoki, Yoshihiro Katayama, Miyuki Sasaki, Yasunori Minakawa, Masao FukudaAbstract:Sphingomonas paucimobilis SYK-6 degrades syringate to 3-O-methylgallate (3MGA), which is finally converted to pyruvate and oxaloacetate via multiple pathways in which protocatechuate 4,5-dioxygenase, 3MGA dioxygenase, and gallate dioxygenase are involved. Here we isolated the syringate O-demethylase gene (desA), which complemented the growth deficiency on syringate of a Tn5 mutant of the SYK-6 derivative strain. The desA gene is located 929 bp downstream of ferA, encoding feruloyl-coenzyme A synthetase, and consists of a 1,386-bp open reading frame encoding a polypeptide with a molecular mass of 50,721 Da. The deduced amino acid sequence of desA showed 26% identity in a 325-amino-acid overlap with that of gcvT of Escherichia coli, which encodes the tetrahydrofolate (H4folate)-dependent aminomethyltransferase involved in glycine cleavage. The cell extract of E. coli carrying desA converted syringate to 3MGA only when H4folate was added to the reaction mixture. DesA catalyzes the transfer of the methyl moiety of syringate to H4folate, forming 5-methyl-H4folate. Vanillate and 3MGA were also used as substrates for DesA; however, the relative activities toward them were 3 and 0.4% of that toward syringate, respectively. Disruption of desA in SYK-6 resulted in a growth defect on syringate but did not affect growth on vanillate, indicating that desA is essential to syringate degradation. In a previous study the ligH gene, which complements the growth deficiency on vanillate and syringate of a chemical-induced mutant of SYK-6, DC-49, was isolated (S. Nishikawa, T. Sonoki, T. Kasahara, T. Obi, S. Kubota, S. Kawai, N. Morohoshi, and Y. Katayama, Appl. Environ. Microbiol. 64:836-842, 1998). Disruption of ligH resulted in the same phenotype as DC-49; its cell extract, however, was found to be able to convert vanillate and syringate in the presence of H4folate. The possible role of ligH is discussed.