scholarly journals Roles of two glutathione S-transferases in the final step of the β-aryl ether cleavage pathway in Sphingobium sp. strain SYK-6

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yudai Higuchi ◽  
Daisuke Sato ◽  
Naofumi Kamimura ◽  
Eiji Masai
Keyword(s):  
Carbon Atom ◽  
Aromatic Compounds ◽  
Specific Activity ◽  
Gene Products ◽  
Enzyme Properties ◽  
Aryl Ether ◽  
Ether Cleavage ◽  
Glutathione S Transferases

AbstractSphingobium sp. strain SYK-6 is an alphaproteobacterial degrader of lignin-derived aromatic compounds, which can degrade all the stereoisomers of β-aryl ether-type compounds. SYK-6 cells convert four stereoisomers of guaiacylglycerol-β-guaiacyl ether (GGE) into two enantiomers of α-(2-methoxyphenoxy)-β-hydroxypropiovanillone (MPHPV) through GGE α-carbon atom oxidation by stereoselective Cα-dehydrogenases encoded by ligD, ligL, and ligN. The ether linkages of the resulting MPHPV enantiomers are cleaved by stereoselective glutathione (GSH) S-transferases (GSTs) encoded by ligF, ligE, and ligP, generating (βR/βS)-α-glutathionyl-β-hydroxypropiovanillone (GS-HPV) and guaiacol. To date, it has been shown that the gene products of ligG and SLG_04120 (ligQ), both encoding GST, catalyze GSH removal from (βR/βS)-GS-HPV, forming achiral β-hydroxypropiovanillone. In this study, we verified the enzyme properties of LigG and LigQ and elucidated their roles in β-aryl ether catabolism. Purified LigG showed an approximately 300-fold higher specific activity for (βR)-GS-HPV than that for (βS)-GS-HPV, whereas purified LigQ showed an approximately six-fold higher specific activity for (βS)-GS-HPV than that for (βR)-GS-HPV. Analyses of mutants of ligG, ligQ, and both genes revealed that SYK-6 converted (βR)-GS-HPV using both LigG and LigQ, whereas only LigQ was involved in converting (βS)-GS-HPV. Furthermore, the disruption of both ligG and ligQ was observed to lead to the loss of the capability of SYK-6 to convert MPHPV. This suggests that GSH removal from GS-HPV catalyzed by LigG and LigQ, is essential for cellular GSH recycling during β-aryl ether catabolism.

scholarly journals Roles of two glutathione S-transferases in the final step of the β-aryl ether cleavage pathway in Sphingobium sp. strain SYK-6

2020 ◽  
Author(s):  
Yudai Higuchi ◽  
Daisuke Sato ◽  
Naofumi Kamimura ◽  
Eiji Masai
Keyword(s):  
Aromatic Compounds ◽  
Specific Activity ◽  
Value Added ◽  
Degradation Process ◽  
Lignin Biodegradation ◽  
Aryl Ether ◽  
Ether Linkage ◽  
Ether Cleavage ◽  
Subsequent Degradation ◽  
Glutathione S Transferases

ABSTRACTSphingobium sp. strain SYK-6 is an alphaproteobacterial degrader of lignin-derived aromatic compounds, which can degrade all the stereoisomers of β-aryl ether-type compounds. SYK-6 cells convert four stereoisomers of guaiacylglycerol-β-guaiacyl ether (GGE) into two enantiomers of α-(2-methoxyphenoxy)-β-hydroxypropiovanillone (MPHPV) through GGE α-carbon atom oxidation by stereoselective Cα-dehydrogenases encoded by ligD, ligL, and ligN. The ether linkages of the resulting MPHPV enantiomers are cleaved by stereoselective glutathione S-transferases (GSTs) encoded by ligF, ligE, and ligP, generating (βRβS)-α-glutathionyl-β-hydroxypropiovanillone (GS-HPV) and guaiacol. To date, it has been shown that the gene products of ligG and SLG_04120 (ligQ), both encoding GST, catalyze glutathione removal from (βRβS)-GS-HPV, forming achiral β-hydroxypropiovanillone. In this study, we characterized the enzyme properties of LigG and LigQ and elucidated their roles in β-aryl ether catabolism. Purified LigG showed an approximately 300-fold higher specific activity for (βR)-GS-HPV than that for (βS)-GS-HPV, whereas purified LigQ showed an approximately six-fold higher specific activity for (βS)-GS-HPV than that for (βR)-GS-HPV. Analyses of mutants of ligG, ligQ, and both genes revealed that SYK-6 converted (βR)-GS-HPV using both LigG and LigQ, whereas only LigQ was involved in converting (βS)-GS-HPV. Furthermore, the disruption of both ligG and ligQ was observed to lead to the loss of the capability of SYK-6 to convert MPHPV. This suggests that GSH removal from GS-HPV catalyzed by LigG and LigQ, is essential for cellular GSH recycling during β-aryl ether catabolism.IMPORTANCEThe β-aryl ether linkage is most abundant in lignin, comprising 45%–62% of all intermonomer linkages in lignin; thus, cleavage of the β-aryl ether linkage together with the subsequent degradation process is considered the essential step in lignin biodegradation. The enzyme genes for β-aryl ether cleavage are useful for decomposing high-molecular-weight lignin, converting lignin-derived aromatic compounds into value-added products, and modifying lignin structures in plants to reduce lignin recalcitrance. In this study, we uncovered the roles of the two glutathione S-transferase genes, ligG and ligQ, in the conversion of GS-HPV isomers, which are generated in the β-aryl ether cleavage pathway in SYK-6. Adding our current results to previous findings allowed us to have a whole picture of the β-aryl ether cleavage system in SYK-6.

Regioselective hydrogenolysis of aryl ether C–O bonds by tungsten carbides with controlled phase compositions

2017 ◽  
Vol 53 (74) ◽  
pp. 10295-10298 ◽  
Author(s):  
Huihuang Fang ◽  
Junmou Du ◽  
Chenchen Tian ◽  
Jianwei Zheng ◽  
Xinping Duan ◽  
...  
Keyword(s):  
Aromatic Compounds ◽  
Aryl Ether ◽  
Tungsten Carbides

Evenly dispersed tungsten carbides with controlled phase compositions that exhibit an impressive capacity to carry out the regioselective hydrogenolysis of inert aryl ether C–O bonds instead of aliphatic C–O bonds to produce aromatic compounds are reported.

scholarly journals Characterization of glutathione S-transferases in rat kidney. Alteration of composition by cis-platinum

1988 ◽  
Vol 252 (1) ◽  
pp. 127-136 ◽  
Author(s):  
G M Trakshel ◽  
M D Maines
Keyword(s):  
Specific Activity ◽  
Rat Kidney ◽  
Male Rats ◽  
Glutathione S Transferase ◽  
Aberrant Form ◽  
Glutathione S Transferases ◽  
Exchange Matrix ◽  
Rat Kidney Cytosol

We have developed chromatographic and mathematical protocols that allowed the high resolution of glutathione S-transferase (GST) subunits, and the identification of a previously unresolved GST monomer in rat kidney cytosol; the monomer was identified tentatively as subunit 6. Also, an aberrant form of GST 7-7 dimer appeared to be present in the kidney. This development was utilized to illustrate the response of rat kidney GST following cis-platinum treatment in vivo. Rat kidney cytosol was separated into three ‘affinity families’ of GST activity after elution from a GSH-agarose matrix. The affinity peaks were characterized by quantitative differences in their subunit and dimeric compositions as determined by subsequent chromatography on a cation-exchange matrix and specific activity towards substrates. By use of these criteria, the major GST dimers of affinity peaks were tentatively identified. The major GST dimers in peak I were GST 1-1 and 1-2, in affinity peak II it was GST 2-2, and in peak III they were GST 3-3 and 7-7. GST 3-6 and/or 4-6, which have not been previously resolved in kidney cytosol, were also present in peak II. Alterations in the kidney cytosolic GST composition of male rats were detected subsequent to the administration of cis-platinum (7.0 mg/kg subcutaneously, 6 days). This treatment caused a pronounced alteration in the GST profile, and the pattern of alteration was markedly different from that reported for other chemicals in the kidney or in the liver. In general, the cellular contents of the GSTs of the Alpha and the Mu classes decreased and increased respectively. It is postulated that the decrease in the Alpha class of GSTs by cis-platinum treatment may be related to renal cortical damage and the loss of GSTs in the urine. The increase in the Mu class of GSTs could potentially stem from a lowered serum concentration of testosterone; the latter is a known effect of cis-platinum treatment.

scholarly journals Cloning and expression of a chick liver glutathione S-transferase CL 3 subunit with the use of a baculovirus expression system

1992 ◽  
Vol 281 (2) ◽  
pp. 545-551 ◽  
Author(s):  
L H Chang ◽  
J Y Fan ◽  
L F Liu ◽  
S P Tsai ◽  
M F Tam
Keyword(s):  
Specific Activity ◽  
Sequence Similarity ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Relative Activity ◽  
Cloning And Expression ◽  
Glutathione S Transferase ◽  
Sds Page ◽  
Glutathione S Transferases ◽  
A Subunit

Glutathione S-transferase CL 3 subunits purified from 1-day-old-chick livers were digested with Achromobacter proteinase I and the resulting fragments were isolated for amino acid sequence analysis. An oligonucleotide probe was constructed accordingly for cDNA library screening. A cDNA clone of 1342 bases, pGCL301, encoding a protein of 26209 Da was isolated and sequenced. Including conservative substitutions, this protein has 75-79% sequence similarity to other Alpha family glutathione S-transferases. The coding sequence of pGCL301 was inserted into a baculovirus vector for infection of Spodoptera frugiperda (SF9) cells. The expressed protein has a high relative activity with ethacrynic acid (47% of the specific activity with 1-chloro-2,4-dinitrobenzene). The enzyme has a subunit molecular mass of 25.2 +/- 1.2 kDa (by SDS/PAGE), a pI of 9.45 and an absorption coefficient A1%1cm of 13.0 +/- 0.5 at 280 nm.

scholarly journals Structural and Biochemical Characterization of the Early and Late Enzymes in the Lignin β-Aryl Ether Cleavage Pathway fromSphingobiumsp. SYK-6

2016 ◽  
Vol 291 (19) ◽  
pp. 10228-10238 ◽  
Author(s):  
Jose Henrique Pereira ◽  
Richard A. Heins ◽  
Daniel L. Gall ◽  
Ryan P. McAndrew ◽  
Kai Deng ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Aryl Ether ◽  
Ether Cleavage

Thermostable esterase estUT1 of bacteria Ureibacillus thermosphaericus: the influence of additional processed domain TrxA on enzyme properties

2019 ◽  
Vol 19 (5) ◽  
pp. 399-407
Author(s):  
K. N. Sorokina ◽  
Yu. V. Samoilova ◽  
V. N. Parmon
Keyword(s):  
Specific Activity ◽  
Enzyme Properties ◽  
Ph Optimum ◽  
T7 Promoter ◽  
Genetic Construct ◽  
E Coli ◽  
Additional Domain ◽  
Ureibacillus Thermosphaericus ◽  
The Stability ◽  
Dissolved Form

A gene of thermostable esterase of bacteria Ureibacillus thermosphaericus was expressed in strain E. coli BL21(DE3) comprised in domain TrxA-containing genetic construct pET32b-estUT1, under the control T7-promoter. The specific activity and relative thermostability of thus produced recombinant enzyme increased from 54.2 to 65.8 % (an hour incubation at 70 °C). The additional domain TrxA was discovered not to affect noticeably the pH optimum of the enzyme activity and its substrate specificity. In the absence of domain TrxA, the stability of estUT1 increased considerably in the presence of various chemicals including ethanol and methanol. The maximal catalytic activity (kcat/KM) of esterase equal to 280.0 s–1·mM–1 was observed in the absence of domain TrxA. Thus, introduction of an additional processed domain TrxA allows the enzyme to be secreted in the dissolved form but, on the other hand, the target protein becomes less thermostable.

scholarly journals Bacterial Catabolism of β-Hydroxypropiovanillone and β-Hydroxypropiosyringone Produced in the Reductive Cleavage of Arylglycerol-β-Aryl Ether in Lignin

2018 ◽  
Vol 84 (7) ◽  
Author(s):  
Yudai Higuchi ◽  
Shogo Aoki ◽  
Hiroki Takenami ◽  
Naofumi Kamimura ◽  
Kenji Takahashi ◽  
...  
Keyword(s):  
Value Added ◽  
Lignin Biodegradation ◽  
Gene Products ◽  
Aryl Ether ◽  
Ether Linkage ◽  
Content Type ◽  
Catabolic Genes ◽  
Genes Encoding ◽  
Stereospecific Reaction

ABSTRACTSphingobiumsp. strain SYK-6 converts four stereoisomers of arylglycerol-β-guaiacyl ether into achiral β-hydroxypropiovanillone (HPV) via three stereospecific reaction steps. Here, we determined the HPV catabolic pathway and characterized the HPV catabolic genes involved in the first two steps of the pathway. In SYK-6 cells, HPV was oxidized to vanilloyl acetic acid (VAA) via vanilloyl acetaldehyde (VAL). The resulting VAA was further converted into vanillate through the activation of VAA by coenzyme A. A syringyl-type HPV analog, β-hydroxypropiosyringone (HPS), was also catabolized via the same pathway. SLG_12830 (hpvZ), which belongs to the glucose-methanol-choline oxidoreductase family, was isolated as the HPV-converting enzyme gene. AnhpvZmutant completely lost the ability to convert HPV and HPS, indicating thathpvZis essential for the conversion of both the substrates. HpvZ produced inEscherichia colioxidized both HPV and HPS and other 3-phenyl-1-propanol derivatives. HpvZ localized to both the cytoplasm and membrane of SYK-6 and used ubiquinone derivatives as electron acceptors. Thirteen gene products of the 23 aldehyde dehydrogenase (ALDH) genes in SYK-6 were able to oxidize VAL into VAA. Mutant analyses suggested that multiple ALDH genes, including SLG_20400, contribute to the conversion of VAL. We examined whether the genes encoding feruloyl-CoA synthetase (ferA) and feruloyl-CoA hydratase/lyase (ferBandferB2) are involved in the conversion of VAA. Only FerA exhibited activity toward VAA; however, disruption offerAdid not affect VAA conversion. These results indicate that another enzyme system is involved in VAA conversion.IMPORTANCECleavage of the β-aryl ether linkage is the most essential process in lignin biodegradation. Although the bacterial β-aryl ether cleavage pathway and catabolic genes have been well documented, there have been no reports regarding the catabolism of HPV or HPS, the products of cleavage of β-aryl ether compounds. HPV and HPS have also been found to be obtained from lignin by chemoselective catalytic oxidation by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone/tert-butyl nitrite/O2, followed by cleavage of the β-aryl ether with zinc. Therefore, value-added chemicals are expected to be produced from these compounds. In this study, we determined the SYK-6 catabolic pathways for HPV and HPS and identified the catabolic genes involved in the first two steps of the pathways. Since SYK-6 catabolizes HPV through 2-pyrone-4,6-dicarboxylate, which is a building block for functional polymers, characterization of HPV catabolism is important not only for understanding the bacterial lignin catabolic system but also for lignin utilization.

scholarly journals Roles of the Enantioselective Glutathione S-Transferases in Cleavage of β-Aryl Ether

2003 ◽  
Vol 185 (6) ◽  
pp. 1768-1775 ◽  
Author(s):  
Eiji Masai ◽  
Atsushi Ichimura ◽  
Yusuke Sato ◽  
Keisuke Miyauchi ◽  
Yoshihiro Katayama ◽  
...  
Keyword(s):  
Lignin Degradation ◽  
Model Compound ◽  
Nucleophilic Attack ◽  
Low Molecular Weight ◽  
Ionization Mass ◽  
Important Process ◽  
Aryl Ether ◽  
Ether Linkage ◽  
Glutathione S Transferases ◽  
Alternative Activities

ABSTRACT Cleavage of the β-aryl ether linkage is the most important process in lignin degradation. Here we characterize the three tandemly located glutathione S-transferase (GST) genes, ligF, ligE, and ligG, from low-molecular-weight lignin-degrading Sphingomonas paucimobilis SYK-6, and we describe the actual roles of these genes in the β-aryl ether cleavage. Based on the identification of the reaction product by electrospray ionization-mass spectrometry, a model compound of β-aryl ether, α-(2-methoxyphenoxy)-β-hydroxypropiovanillone (MPHPV), was transformed by LigF or LigE to guaiacol and α-glutathionyl-β-hydroxypropiovanillone (GS-HPV). This result suggested that LigF and LigE catalyze the nucleophilic attack of glutathione on the carbon atom at the β position of MPHPV. High-pressure liquid chromatography-circular dichroism analysis indicated that LigF and LigE each attacked a different enantiomer of the racemic MPHPV preparation. The ligG gene product specifically catalyzed the elimination of glutathione from GS-HPV generated by the action of LigF. This reaction then produces an achiral compound, β-hydroxypropiovanillone, which is further degraded by this strain. Disruption of the ligF, ligE, and ligG genes in SYK-6 showed that ligF is essential to the degradation of one of the MPHPV enantiomers, and the alternative activities which metabolize the substrates of LigE and LigG are present in this strain.

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