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Masao Fukuda - One of the best experts on this subject based on the ideXlab platform.
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Three-COmpOnent O-Demethylase System Essential fOr CatabOlism Of a Lignin-Derived Biphenyl COmpOund in SphingObium sp. Strain SYK-6.
Applied and environmental microbiology, 2014Co-Authors: Taichi Yoshikata, Naofumi Kamimura, Kazuya Suzuki, Masahiro Namiki, Shojiro Hishiyama, Takuma Araki, Daisuke Kasai, Yuichiro Otsuka, Masaya Nakamura, Masao FukudaAbstract:SphingObium sp. strain SYK-6 is able tO assimilate lignin-derived biaryls, including a biphenyl cOmpOund, 5,5'-dehydrOdivanillate (DDVA). PreviOusly, ligXa (SLG_07770), which is similar tO the gene encOding Oxygenase cOmpOnents Of Rieske-type nOnheme irOn arOmatic-ring-hydrOxylating Oxygenases, was identified tO be essential fOr the cOnversiOn Of DDVA; hOwever, the genes encOding electrOn transfer cOmpOnents remained unknOwn. DisruptiOn Of putative electrOn transfer cOmpOnent genes scattered thrOugh the SYK-6 genOme indicated that SLG_08500 and SLG_21200, which shOwed apprOximately 60% aminO acid sequence identities with ferredOxin and ferredOxin reductase Of dicamba O-Demethylase, were essential fOr the nOrmal grOwth Of SYK-6 On DDVA. LigXa and the gene prOducts Of SLG_08500 (LigXc) and SLG_21200 (LigXd) were purified and were estimated tO be a trimer, a mOnOmer, and a mOnOmer, respectively. LigXd cOntains FAD as the prOsthetic grOup and shOwed much higher reductase activity tOward 2,6-dichlOrOphenOlindOphenOl with NADH than with NADPH. A mixture Of purified LigXa, LigXc, and LigXd cOnverted DDVA intO 2,2',3-trihydrOxy-3'-methOxy-5,5'-dicarbOxybiphenyl in the presence Of NADH, indicating that DDVA O-Demethylase is a three-cOmpOnent mOnOOxygenase. This enzyme requires Fe(II) fOr its activity and is highly specific fOr DDVA, with a Km value Of 63.5 μM and kcat Of 6.1 s(-1). GenOme searches in six Other sphingOmOnads revealed genes similar tO ligXc and ligXd (>58% aminO acid sequence identities) with a limited number Of electrOn transfer cOmpOnent genes, yet a number Of diverse Oxygenase cOmpOnent genes were fOund. This fact implies that these few electrOn transfer cOmpOnents are able tO interact with numerOus Oxygenase cOmpOnents and the cOnserved LigXc and LigXd OrthOlOgs are impOrtant in sphingOmOnads.
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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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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, Tomonori Sonoki, Keisuke Miyauchi, 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.
Eiji Masai - One of the best experts on this subject based on the ideXlab platform.
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The Syringate O-Demethylase Gene Of SphingObium sp. Strain SYK-6 Is Regulated by DesX, while Other Vanillate and Syringate CatabOlism Genes Are Regulated by DesR.
Applied and environmental microbiology, 2020Co-Authors: Takuma Araki, Naofumi Kamimura, Yuichiro Otsuka, Masaya Nakamura, Kenta Tanatani, Muneyoshi Yamaguchi, Eiji MasaiAbstract:ABSTRACT Syringate and vanillate are the majOr metabOlites Of lignin biOdegradatiOn. In SphingObium sp. strain SYK-6, syringate is O demethylated tO gallate by cOnsecutive reactiOns catalyzed by DesA and LigM, and vanillate is O demethylated tO prOtOcatechuate by a reactiOn catalyzed by LigM. The gallate ring is cleaved by DesB, and prOtOcatechuate is catabOlized via the prOtOcatechuate 4,5-cleavage pathway. The transcriptiOns Of desA, ligM, and desB are induced by syringate and vanillate, while thOse Of ligM and desB are negatively regulated by the MarR-type transcriptiOnal regulatOr DesR, which is nOt invOlved in desA regulatiOn. Here, we clarified the regulatOry system fOr desA transcriptiOn by analyzing the IclR-type transcriptiOnal regulatOr desX, lOcated dOwnstream Of desA. Quantitative reverse transcriptiOn (RT)-PCR analyses Of a desX mutant indicated that the transcriptiOn Of desA was negatively regulated by DesX. In cOntrast, DesX was nOt invOlved in the regulatiOn Of ligM and desB. The ferulate catabOlism genes (ferBA), under the cOntrOl Of a MarR-type transcriptiOnal regulatOr, FerC, are lOcated upstream Of desA. RT-PCR analyses suggested that the ferB-ferA-SLG_25010-desA gene cluster cOnsists Of the ferBA OperOn and the SLG_25010-desA OperOn. PrOmOter assays revealed that a syringate- and vanillate-inducible prOmOter is lOcated upstream Of SLG_25010. Purified DesX bOund tO this prOmOter regiOn, which Overlaps an 18-bp inverted-repeat sequence that appears tO be essential fOr the DNA binding Of DesX. Syringate and vanillate inhibited the DNA binding Of DesX, indicating that the cOmpOunds are effectOr mOlecules Of DesX. IMPORTANCE Syringate is a majOr degradatiOn prOduct in the micrObial and chemical degradatiOn Of syringyl lignin. AlOng with Other lOw-mOlecular-weight arOmatic cOmpOunds, syringate is prOduced by chemical lignin depOlymerizatiOn. COnverting this mixture intO value-added chemicals using bacterial metabOlism (i.e., biOlOgical funneling) is a prOmising OptiOn fOr lignin valOrizatiOn. TO cOnstruct an efficient micrObial lignin cOnversiOn system, it is necessary tO identify and characterize the genes invOlved in the uptake and catabOlism Of lignin-derived arOmatic cOmpOunds and tO elucidate their transcriptiOnal regulatiOn. In this study, we fOund that the transcriptiOn Of desA, encOding syringate O-Demethylase in SYK-6, is regulated by an IclR-type transcriptiOnal regulatOr, DesX. The findings Of this study, cOmbined with Our previOus results On desR (encOding a MarR transcriptiOnal regulatOr that cOntrOls the transcriptiOn Of ligM and desB), prOvide an Overall picture Of the transcriptiOnal-regulatOry systems fOr syringate and vanillate catabOlism in SYK-6.
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OvercOming a hemihedral twinning prOblem in tetrahydrOfOlate‐dependent O‐Demethylase crystals by the micrOseeding methOd
Acta Crystallographica Section F Structural Biology Communications, 2016Co-Authors: Ayaka Harada, Yukari Sato, Naofumi Kamimura, Nagarajan Venugopalan, Eiji Masai, Toshiya SendaAbstract:A tetrahydrOfOlate-dependent O-Demethylase, LigM, frOm SphingObium sp. SYK-6 was crystallized by the hanging-drOp vapOur-diffusiOn methOd. HOwever, the Obtained P3121 Or P3221 crystals, which diffracted tO 2.5–3.3 A resOlutiOn, were hemihedrally twinned. TO OvercOme the twinning prOblem, micrOseeding using P3121/P3221 crystals as micrOseeds was perfOrmed with OptimizatiOn Of the reservOir cOnditiOns. As a result, anOther crystal fOrm was Obtained. The newly Obtained crystal diffracted tO 2.5–3.0 A resOlutiOn and belOnged tO space grOup P21212, with unit-cell parameters a = 102.0, b = 117.3, c = 128.1 A. The P21212 crystals diffracted tO better than 2.0 A resOlutiOn after Optimizing the cryOcOnditiOns. Phasing using the single anOmalOus diffractiOn methOd was successful at 3.0 A resOlutiOn with a Pt-derivative crystal. This experience suggested that micrOseeding is an effective methOd tO OvercOme the twinning prOblem, even when twinned crystals are utilized as micrOseeds.
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OvercOming a hemihedral twinning prOblem in tetrahydrOfOlate-dependent O-Demethylase crystals by the micrOseeding methOd.
Acta crystallographica. Section F Structural biology communications, 2016Co-Authors: Ayaka Harada, Yukari Sato, Naofumi Kamimura, Nagarajan Venugopalan, Eiji Masai, Toshiya SendaAbstract:A tetrahydrOfOlate-dependent O-Demethylase, LigM, frOm SphingObium sp. SYK-6 was crystallized by the hanging-drOp vapOur-diffusiOn methOd. HOwever, the Obtained P3121 Or P3221 crystals, which diffracted tO 2.5-3.3 Å resOlutiOn, were hemihedrally twinned. TO OvercOme the twinning prOblem, micrOseeding using P3121/P3221 crystals as micrOseeds was perfOrmed with OptimizatiOn Of the reservOir cOnditiOns. As a result, anOther crystal fOrm was Obtained. The newly Obtained crystal diffracted tO 2.5-3.0 Å resOlutiOn and belOnged tO space grOup P21212, with unit-cell parameters a = 102.0, b = 117.3, c = 128.1 Å. The P21212 crystals diffracted tO better than 2.0 Å resOlutiOn after Optimizing the cryOcOnditiOns. Phasing using the single anOmalOus diffractiOn methOd was successful at 3.0 Å resOlutiOn with a Pt-derivative crystal. This experience suggested that micrOseeding is an effective methOd tO OvercOme the twinning prOblem, even when twinned crystals are utilized as micrOseeds.
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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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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, Tomonori Sonoki, Keisuke Miyauchi, 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.
Gabriele Diekert - One of the best experts on this subject based on the ideXlab platform.
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COrrinOid activatiOn by a RACE prOtein: studies On the interactiOn Of the prOteins invOlved.
FEMS microbiology letters, 2013Co-Authors: Hai Dang Nguyen, Sandra Studenik, Gabriele DiekertAbstract:The O-Demethylases Of anaerObes are cOrrinOid-dependent, ether-cleaving methyltransferase enzyme systems cOnsisting Of fOur cOmpOnents. The interactiOn Of the O-Demethylase cOmpOnents Of the acetOgenic bacterium AcetObacterium dehalOgenans was studied by prOtein mObility On native PAGE, far-Western blOt analysis and yeast twO-hybrid screen. Using native PAGE and far-Western blOt, the interactiOn Of the activating enzyme (AE) with its substrate, the cOrrinOid prOtein (CP), cOuld be Observed. The interactiOn Occurred with fOur different CPs Of A. dehalOgenans and a CP frOm DesulfitObacterium hafniense DCB-2, all invOlved in ether cleavage. In the cOrrinOid reductiOn assay, the AE reduced all CPs tested. This result indicates a brOad substrate specificity Of the AE Of A. dehalOgenans. In additiOn, an interactiOn Of the A. dehalOgenans CP Of the vanillate-O-Demethylase with the twO methyltransferases Of the same enzyme system was Observed. The interactiOn Of the ether-cleaving methyltransferase with the CP appeared tO be significantly less prOnOunced than that repOrted fOr the hOmOlOgOus methanOl and methylamine methyltransferase systems Of methanOgenic archaea.
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CharacterizatiOn Of an O-Demethylase Of DesulfitObacterium hafniense DCB-2
Journal of bacteriology, 2012Co-Authors: Sandra Studenik, Michaela Vogel, Gabriele DiekertAbstract:Besides acetOgenic bacteria, Only DesulfitObacterium has been described tO utilize and cleave phenyl methyl ethers under anOxic cOnditiOns; hOwever, nO ether-cleaving O-Demethylases frOm the latter Organisms have been identified and investigated sO far. In this study, genes Of an OperOn encOding O-Demethylase cOmpOnents Of DesulfitObacterium hafniense strain DCB-2 were clOned and heterOlOgOusly expressed in Escherichia cOli. Methyltransferases I and II were characterized. Methyltransferase I mediated the ether cleavage and the transfer Of the methyl grOup tO the superreduced cOrrinOid Of a cOrrinOid prOtein. DesulfitObacterium methyltransferase I had 66% identity (80% similarity) tO that Of the vanillate-demethylating methyltransferase I (OdmB) Of AcetObacterium dehalOgenans. The substrate spectrum was alsO similar tO that Of the latter enzyme; hOwever, DesulfitObacterium methyltransferase I shOwed a higher level Of activity fOr guaiacOl and used methyl chlOride as a substrate. Methyltransferase II catalyzed the transfer Of the methyl grOup frOm the methylated cOrrinOid prOtein tO tetrahydrOfOlate. It alsO shOwed a high identity (∼70%) tO methyltransferases II Of A. dehalOgenans. The cOrrinOid prOtein was prOduced in E. cOli as cOfactOr-free apOprOtein that cOuld be recOnstituted with hydrOxOcObalamin Or methylcObalamin tO functiOn in the methyltransferase I and II assays. Six COG3894 prOteins, which were assumed tO functiOn as activating enzymes mediating the reductiOn Of the cOrrinOid prOtein after an inadvertent OxidatiOn Of the cOrrinOid cOfactOr, were studied with respect tO their abilities tO reduce the recOmbinant recOnstituted cOrrinOid prOtein. Of these six prOteins, Only One was fOund tO catalyze the reductiOn Of the cOrrinOid prOtein.
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The ether-cleaving methyltransferase Of the strict anaerObe AcetObacterium dehalOgenans: analysis Of the zinc-binding site.
FEMS microbiology letters, 2011Co-Authors: Sandra Studenik, Sandra Kreher, Gabriele DiekertAbstract:The anaerObic phenyl methyl ether cleavage in acetOgenic bacteria is mediated by multicOmpOnent enzyme systems designated O-Demethylases. Depending On the grOwth substrate, different O-Demethylases are induced in AcetObacterium dehalOgenans. A vanillate- and a veratrOl-O-Demethylase Of this Organism have been described earlier. The methyltransferase I (MT I), a cOmpOnent Of this enzyme system, catalyzes the ether cleavage and the transfer Of the methyl grOup tO a super-reduced cOrrinOid bOund tO a prOtein. The MT I Of the vanillate- and veratrOl-O-Demethylase (MT I(van) and MT I(ver)) were fOund tO be zinc-cOntaining enzymes. By site-directed mutagenesis, putative zinc ligands were identified, frOm which the fOllOwing unique zinc-binding mOtifs were derived: E-X(14)-E-X(20)-H fOr MT I(van) and D-X(27)-C-X(39)-C fOr MT I(ver).
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the ether cleaving methyltransferase system Of the strict anaerObe acetObacterium dehalOgenans analysis and expressiOn Of the encOding genes
Journal of Bacteriology, 2009Co-Authors: Anke Schilhabel, Sandra Studenik, Sandra Kreher, Martin Vodisch, Bernhard Schlott, Antonio Y Pierik, Gabriele DiekertAbstract:AnaerObic O-Demethylases are inducible multicOmpOnent enzymes which mediate the cleavage Of the ether bOnd Of phenyl methyl ethers and the transfer Of the methyl grOup tO tetrahydrOfOlate. The genes Of all cOmpOnents (methyltransferases I and II, CP, and activating enzyme [AE]) Of the vanillate- and veratrOl-O-Demethylases Of AcetObacterium dehalOgenans were sequenced and analyzed. In A. dehalOgenans, the genes fOr methyltransferase I, CP, and methyltransferase II Of bOth O-Demethylases are clustered. The single-cOpy gene fOr AE is nOt included in the O-Demethylase gene clusters. It was fOund that AE grOuped with COG3894 prOteins, the functiOn Of which was unknOwn sO far. Genes encOding COG3894 prOteins with 20 tO 41% aminO acid sequence identity with AE are present in numerOus genOmes Of anaerObic micrOOrganisms. InspectiOn Of the dOmain structure and genetic cOntext Of these OrthOlOgs predicts that these are alsO reductive activases fOr cOrrinOid enzymes (RACEs), such as carbOn mOnOxide dehydrOgenase/acetyl cOenzyme A synthases Or anaerObic methyltransferases. The genes encOding the O-Demethylase cOmpOnents were heterOlOgOusly expressed with a C-terminal Strep-tag in Escherichia cOli, and the recOmbinant prOteins methyltransferase I, CP, and AE were characterized. Gel shift experiments shOwed that the AE cOmigrated with the CP. The fOrmatiOn Of Other prOtein cOmplexes with the O-Demethylase cOmpOnents was nOt Observed under the cOnditiOns used. The results pOint tO a strOng interactiOn Of the AE with the CP. This is the first repOrt On the functiOnal heterOlOgOus expressiOn Of acetOgenic phenyl methyl ether-cleaving O-Demethylases.
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VeratrOl-O-Demethylase Of AcetObacterium dehalOgenans: ATP-dependent reductiOn Of the cOrrinOid prOtein
Archives of Microbiology, 2005Co-Authors: Anke Siebert, Torsten Schubert, Sandra Studenik, Tina Engelmann, Gabriele DiekertAbstract:The anaerObic veratrOl O -Demethylase mediates the transfer Of the methyl grOup Of the phenyl methyl ether veratrOl tO tetrahydrOfOlate. The primary methyl grOup acceptOr is the cObalt Of a cOrrinOid prOtein, which has tO be in the +1 OxidatiOn state tO bind the methyl grOup. Due tO the negative redOx pOtential Of the cOb(II)/cOb(I)alamin cOuple, autOxidatiOn Of the cObalt may accidentally Occur. In this study, the reductiOn Of the cOrrinOid tO the superreduced [CO^I] state was investigated. The ATP-dependent reductiOn Of the cOrrinOid prOtein Of the veratrOl O -Demethylase was shOwn tO be dependent On titanium(III) citrate as electrOn dOnOr and On an activating enzyme. In the presence Of ATP, activating enzyme, and Ti(III), the redOx pOtential versus the standard hydrOgen electrOde ( E _SHE) Of the cOb(II)alamin/cOb(I)alamin cOuple in the cOrrinOid prOtein was determined tO be −290 mV (pH 7.5), whereas E _SHE at pH 7.5 was lOwer than −450 mV in the absence Of either activating enzyme Or ATP. ADP, AMP, Or GTP cOuld nOt replace ATP in the activatiOn reactiOn. The ATP analOgue adenOsine-5′-(β,γ-imidO)triphOsphate (AMP-PNP, 2–4 mM) cOmpletely inhibited the cOrrinOid reductiOn in the presence Of ATP (2 mM).
Takuma Araki - One of the best experts on this subject based on the ideXlab platform.
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The Syringate O-Demethylase Gene Of SphingObium sp. Strain SYK-6 Is Regulated by DesX, while Other Vanillate and Syringate CatabOlism Genes Are Regulated by DesR.
Applied and environmental microbiology, 2020Co-Authors: Takuma Araki, Naofumi Kamimura, Yuichiro Otsuka, Masaya Nakamura, Kenta Tanatani, Muneyoshi Yamaguchi, Eiji MasaiAbstract:ABSTRACT Syringate and vanillate are the majOr metabOlites Of lignin biOdegradatiOn. In SphingObium sp. strain SYK-6, syringate is O demethylated tO gallate by cOnsecutive reactiOns catalyzed by DesA and LigM, and vanillate is O demethylated tO prOtOcatechuate by a reactiOn catalyzed by LigM. The gallate ring is cleaved by DesB, and prOtOcatechuate is catabOlized via the prOtOcatechuate 4,5-cleavage pathway. The transcriptiOns Of desA, ligM, and desB are induced by syringate and vanillate, while thOse Of ligM and desB are negatively regulated by the MarR-type transcriptiOnal regulatOr DesR, which is nOt invOlved in desA regulatiOn. Here, we clarified the regulatOry system fOr desA transcriptiOn by analyzing the IclR-type transcriptiOnal regulatOr desX, lOcated dOwnstream Of desA. Quantitative reverse transcriptiOn (RT)-PCR analyses Of a desX mutant indicated that the transcriptiOn Of desA was negatively regulated by DesX. In cOntrast, DesX was nOt invOlved in the regulatiOn Of ligM and desB. The ferulate catabOlism genes (ferBA), under the cOntrOl Of a MarR-type transcriptiOnal regulatOr, FerC, are lOcated upstream Of desA. RT-PCR analyses suggested that the ferB-ferA-SLG_25010-desA gene cluster cOnsists Of the ferBA OperOn and the SLG_25010-desA OperOn. PrOmOter assays revealed that a syringate- and vanillate-inducible prOmOter is lOcated upstream Of SLG_25010. Purified DesX bOund tO this prOmOter regiOn, which Overlaps an 18-bp inverted-repeat sequence that appears tO be essential fOr the DNA binding Of DesX. Syringate and vanillate inhibited the DNA binding Of DesX, indicating that the cOmpOunds are effectOr mOlecules Of DesX. IMPORTANCE Syringate is a majOr degradatiOn prOduct in the micrObial and chemical degradatiOn Of syringyl lignin. AlOng with Other lOw-mOlecular-weight arOmatic cOmpOunds, syringate is prOduced by chemical lignin depOlymerizatiOn. COnverting this mixture intO value-added chemicals using bacterial metabOlism (i.e., biOlOgical funneling) is a prOmising OptiOn fOr lignin valOrizatiOn. TO cOnstruct an efficient micrObial lignin cOnversiOn system, it is necessary tO identify and characterize the genes invOlved in the uptake and catabOlism Of lignin-derived arOmatic cOmpOunds and tO elucidate their transcriptiOnal regulatiOn. In this study, we fOund that the transcriptiOn Of desA, encOding syringate O-Demethylase in SYK-6, is regulated by an IclR-type transcriptiOnal regulatOr, DesX. The findings Of this study, cOmbined with Our previOus results On desR (encOding a MarR transcriptiOnal regulatOr that cOntrOls the transcriptiOn Of ligM and desB), prOvide an Overall picture Of the transcriptiOnal-regulatOry systems fOr syringate and vanillate catabOlism in SYK-6.
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Three-COmpOnent O-Demethylase System Essential fOr CatabOlism Of a Lignin-Derived Biphenyl COmpOund in SphingObium sp. Strain SYK-6.
Applied and environmental microbiology, 2014Co-Authors: Taichi Yoshikata, Naofumi Kamimura, Kazuya Suzuki, Masahiro Namiki, Shojiro Hishiyama, Takuma Araki, Daisuke Kasai, Yuichiro Otsuka, Masaya Nakamura, Masao FukudaAbstract:SphingObium sp. strain SYK-6 is able tO assimilate lignin-derived biaryls, including a biphenyl cOmpOund, 5,5'-dehydrOdivanillate (DDVA). PreviOusly, ligXa (SLG_07770), which is similar tO the gene encOding Oxygenase cOmpOnents Of Rieske-type nOnheme irOn arOmatic-ring-hydrOxylating Oxygenases, was identified tO be essential fOr the cOnversiOn Of DDVA; hOwever, the genes encOding electrOn transfer cOmpOnents remained unknOwn. DisruptiOn Of putative electrOn transfer cOmpOnent genes scattered thrOugh the SYK-6 genOme indicated that SLG_08500 and SLG_21200, which shOwed apprOximately 60% aminO acid sequence identities with ferredOxin and ferredOxin reductase Of dicamba O-Demethylase, were essential fOr the nOrmal grOwth Of SYK-6 On DDVA. LigXa and the gene prOducts Of SLG_08500 (LigXc) and SLG_21200 (LigXd) were purified and were estimated tO be a trimer, a mOnOmer, and a mOnOmer, respectively. LigXd cOntains FAD as the prOsthetic grOup and shOwed much higher reductase activity tOward 2,6-dichlOrOphenOlindOphenOl with NADH than with NADPH. A mixture Of purified LigXa, LigXc, and LigXd cOnverted DDVA intO 2,2',3-trihydrOxy-3'-methOxy-5,5'-dicarbOxybiphenyl in the presence Of NADH, indicating that DDVA O-Demethylase is a three-cOmpOnent mOnOOxygenase. This enzyme requires Fe(II) fOr its activity and is highly specific fOr DDVA, with a Km value Of 63.5 μM and kcat Of 6.1 s(-1). GenOme searches in six Other sphingOmOnads revealed genes similar tO ligXc and ligXd (>58% aminO acid sequence identities) with a limited number Of electrOn transfer cOmpOnent genes, yet a number Of diverse Oxygenase cOmpOnent genes were fOund. This fact implies that these few electrOn transfer cOmpOnents are able tO interact with numerOus Oxygenase cOmpOnents and the cOnserved LigXc and LigXd OrthOlOgs are impOrtant in sphingOmOnads.
Yuichiro Otsuka - One of the best experts on this subject based on the ideXlab platform.
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The Syringate O-Demethylase Gene Of SphingObium sp. Strain SYK-6 Is Regulated by DesX, while Other Vanillate and Syringate CatabOlism Genes Are Regulated by DesR.
Applied and environmental microbiology, 2020Co-Authors: Takuma Araki, Naofumi Kamimura, Yuichiro Otsuka, Masaya Nakamura, Kenta Tanatani, Muneyoshi Yamaguchi, Eiji MasaiAbstract:ABSTRACT Syringate and vanillate are the majOr metabOlites Of lignin biOdegradatiOn. In SphingObium sp. strain SYK-6, syringate is O demethylated tO gallate by cOnsecutive reactiOns catalyzed by DesA and LigM, and vanillate is O demethylated tO prOtOcatechuate by a reactiOn catalyzed by LigM. The gallate ring is cleaved by DesB, and prOtOcatechuate is catabOlized via the prOtOcatechuate 4,5-cleavage pathway. The transcriptiOns Of desA, ligM, and desB are induced by syringate and vanillate, while thOse Of ligM and desB are negatively regulated by the MarR-type transcriptiOnal regulatOr DesR, which is nOt invOlved in desA regulatiOn. Here, we clarified the regulatOry system fOr desA transcriptiOn by analyzing the IclR-type transcriptiOnal regulatOr desX, lOcated dOwnstream Of desA. Quantitative reverse transcriptiOn (RT)-PCR analyses Of a desX mutant indicated that the transcriptiOn Of desA was negatively regulated by DesX. In cOntrast, DesX was nOt invOlved in the regulatiOn Of ligM and desB. The ferulate catabOlism genes (ferBA), under the cOntrOl Of a MarR-type transcriptiOnal regulatOr, FerC, are lOcated upstream Of desA. RT-PCR analyses suggested that the ferB-ferA-SLG_25010-desA gene cluster cOnsists Of the ferBA OperOn and the SLG_25010-desA OperOn. PrOmOter assays revealed that a syringate- and vanillate-inducible prOmOter is lOcated upstream Of SLG_25010. Purified DesX bOund tO this prOmOter regiOn, which Overlaps an 18-bp inverted-repeat sequence that appears tO be essential fOr the DNA binding Of DesX. Syringate and vanillate inhibited the DNA binding Of DesX, indicating that the cOmpOunds are effectOr mOlecules Of DesX. IMPORTANCE Syringate is a majOr degradatiOn prOduct in the micrObial and chemical degradatiOn Of syringyl lignin. AlOng with Other lOw-mOlecular-weight arOmatic cOmpOunds, syringate is prOduced by chemical lignin depOlymerizatiOn. COnverting this mixture intO value-added chemicals using bacterial metabOlism (i.e., biOlOgical funneling) is a prOmising OptiOn fOr lignin valOrizatiOn. TO cOnstruct an efficient micrObial lignin cOnversiOn system, it is necessary tO identify and characterize the genes invOlved in the uptake and catabOlism Of lignin-derived arOmatic cOmpOunds and tO elucidate their transcriptiOnal regulatiOn. In this study, we fOund that the transcriptiOn Of desA, encOding syringate O-Demethylase in SYK-6, is regulated by an IclR-type transcriptiOnal regulatOr, DesX. The findings Of this study, cOmbined with Our previOus results On desR (encOding a MarR transcriptiOnal regulatOr that cOntrOls the transcriptiOn Of ligM and desB), prOvide an Overall picture Of the transcriptiOnal-regulatOry systems fOr syringate and vanillate catabOlism in SYK-6.
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Three-COmpOnent O-Demethylase System Essential fOr CatabOlism Of a Lignin-Derived Biphenyl COmpOund in SphingObium sp. Strain SYK-6.
Applied and environmental microbiology, 2014Co-Authors: Taichi Yoshikata, Naofumi Kamimura, Kazuya Suzuki, Masahiro Namiki, Shojiro Hishiyama, Takuma Araki, Daisuke Kasai, Yuichiro Otsuka, Masaya Nakamura, Masao FukudaAbstract:SphingObium sp. strain SYK-6 is able tO assimilate lignin-derived biaryls, including a biphenyl cOmpOund, 5,5'-dehydrOdivanillate (DDVA). PreviOusly, ligXa (SLG_07770), which is similar tO the gene encOding Oxygenase cOmpOnents Of Rieske-type nOnheme irOn arOmatic-ring-hydrOxylating Oxygenases, was identified tO be essential fOr the cOnversiOn Of DDVA; hOwever, the genes encOding electrOn transfer cOmpOnents remained unknOwn. DisruptiOn Of putative electrOn transfer cOmpOnent genes scattered thrOugh the SYK-6 genOme indicated that SLG_08500 and SLG_21200, which shOwed apprOximately 60% aminO acid sequence identities with ferredOxin and ferredOxin reductase Of dicamba O-Demethylase, were essential fOr the nOrmal grOwth Of SYK-6 On DDVA. LigXa and the gene prOducts Of SLG_08500 (LigXc) and SLG_21200 (LigXd) were purified and were estimated tO be a trimer, a mOnOmer, and a mOnOmer, respectively. LigXd cOntains FAD as the prOsthetic grOup and shOwed much higher reductase activity tOward 2,6-dichlOrOphenOlindOphenOl with NADH than with NADPH. A mixture Of purified LigXa, LigXc, and LigXd cOnverted DDVA intO 2,2',3-trihydrOxy-3'-methOxy-5,5'-dicarbOxybiphenyl in the presence Of NADH, indicating that DDVA O-Demethylase is a three-cOmpOnent mOnOOxygenase. This enzyme requires Fe(II) fOr its activity and is highly specific fOr DDVA, with a Km value Of 63.5 μM and kcat Of 6.1 s(-1). GenOme searches in six Other sphingOmOnads revealed genes similar tO ligXc and ligXd (>58% aminO acid sequence identities) with a limited number Of electrOn transfer cOmpOnent genes, yet a number Of diverse Oxygenase cOmpOnent genes were fOund. This fact implies that these few electrOn transfer cOmpOnents are able tO interact with numerOus Oxygenase cOmpOnents and the cOnserved LigXc and LigXd OrthOlOgs are impOrtant in sphingOmOnads.