scholarly journals Three-ComponentO-Demethylase System Essential for Catabolism of a Lignin-Derived Biphenyl Compound in Sphingobium sp. Strain SYK-6

2014 ◽  
Vol 80 (23) ◽  
pp. 7142-7153 ◽  
Author(s):  
Taichi Yoshikata ◽  
Kazuya Suzuki ◽  
Naofumi Kamimura ◽  
Masahiro Namiki ◽  
Shojiro Hishiyama ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Reductase Activity ◽  
Normal Growth ◽  
Prosthetic Group ◽  
Gene Encoding ◽  
Content Type ◽  
Sequence Identities ◽  
Electron Transfer Component

ABSTRACTSphingobiumsp. 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 dicambaO-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 DDVAO-demethylase is a three-component monooxygenase. This enzyme requires Fe(II) for its activity and is highly specific for DDVA, with aKmvalue of 63.5 μM andkcatof 6.1 s−1. Genome searches in six other sphingomonads revealed genes similar toligXcandligXd(>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.

scholarly journals Novel Three-Component Rieske Non-Heme Iron Oxygenase System Catalyzing theN-Dealkylation of Chloroacetanilide Herbicides in Sphingomonads DC-6 and DC-2

2014 ◽  
Vol 80 (16) ◽  
pp. 5078-5085 ◽  
Author(s):  
Qing Chen ◽  
Cheng-Hong Wang ◽  
Shi-Kai Deng ◽  
Ya-Dong Wu ◽  
Yi Li ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Heme Iron ◽  
Content Type ◽  
Non Heme Iron ◽  
Transport Component ◽  
Reductase Gene ◽  
Chloroacetanilide Herbicides ◽  
The Individual ◽  
Sequence Identities

ABSTRACTSphingomonads DC-6 and DC-2 degrade the chloroacetanilide herbicides alachlor, acetochlor, and butachlor viaN-dealkylation. In this study, we report a three-component Rieske non-heme iron oxygenase (RHO) system catalyzing theN-dealkylation of these herbicides. The oxygenase component genecndAis located in a transposable element that is highly conserved in the two strains. CndA shares 24 to 42% amino acid sequence identities with the oxygenase components of some RHOs that catalyzeN- orO-demethylation. Two putative [2Fe-2S] ferredoxin genes and one glutathione reductase (GR)-type reductase gene were retrieved from the genome of each strain. These genes were not located in the immediate vicinity ofcndA. The four ferredoxins share 64 to 72% amino acid sequence identities to the ferredoxin component of dicambaO-demethylase (DMO), and the two reductases share 62 to 65% amino acid sequence identities to the reductase component of DMO.cndA, the four ferredoxin genes, and the two reductases genes were expressed inEscherichia coli, and the recombinant proteins were purified using Ni-affinity chromatography. The individual components or the components in pairs displayed no activity; the enzyme mixture showedN-dealkylase activities toward alachlor, acetochlor, and butachlor only when CndA-His6was combined with one of the four ferredoxins and one of the two reductases, suggesting that the enzyme consists of three components, a homo-oligomer oxygenase, a [2Fe-2S] ferredoxin, and a GR-type reductase, and CndA has a low specificity for the electron transport component (ETC). TheN-dealkylase utilizes NADH, but not NADPH, as the electron donor.

scholarly journals Novel Maltogenic Amylase CoMA fromCorallococcussp. Strain EGB Catalyzes the Conversion of Maltooligosaccharides and Soluble Starch to Maltose

2018 ◽  
Vol 84 (14) ◽  
Author(s):  
Jie Zhou ◽  
Zhoukun Li ◽  
Han Zhang ◽  
Jiale Wu ◽  
Xianfeng Ye ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Biochemical Characterization ◽  
Soluble Starch ◽  
Glycoside Hydrolase Family ◽  
Gene Encoding ◽  
Commercial Development ◽  
Amylolytic Enzyme ◽  
Content Type ◽  
High Level

ABSTRACTThe gene encoding the novel amylolytic enzyme designated CoMA was cloned fromCorallococcussp. strain EGB. The deduced amino acid sequence contained a predicted lipoprotein signal peptide (residues 1 to 18) and a conserved glycoside hydrolase family 13 (GH13) module. The amino acid sequence of CoMA exhibits low sequence identity (10 to 19%) with cyclodextrin-hydrolyzing enzymes (GH13_20) and is assigned to GH13_36. The most outstanding feature of CoMA is its ability to catalyze the conversion of maltooligosaccharides (≥G3) and soluble starch to maltose as the sole hydrolysate. Moreover, it can hydrolyze γ-cyclodextrin and starch to maltose and hydrolyze pullulan exclusively to panose with relative activities of 0.2, 1, and 0.14, respectively. CoMA showed both hydrolysis and transglycosylation activities toward α-1,4-glycosidic bonds but not to α-1,6-linkages. Moreover, glucosyl transfer was postulated to be the major transglycosidation reaction for producing a high level of maltose without the attendant production of glucose. These results indicated that CoMA possesses some unusual properties that distinguish it from maltogenic amylases and typical α-amylases. Its physicochemical properties suggested that it has potential for commercial development.IMPORTANCEThe α-amylase fromCorallococcussp. EGB, which was classified to the GH13_36 subfamily, can catalyze the conversion of maltooligosaccharides (≥G3) and soluble starch to maltose as the sole hydrolysate. An action mechanism for producing a high level of maltose without the attendant production of glucose has been proposed. Moreover, it also can hydrolyze γ-cyclodextrin and pullulan. Its biochemical characterization suggested that CoMA may be involved the accumulation of maltose inCorallococcusmedia.

Identification of the gene encoding 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid desulfhydrase in Burkholderia sp. HME13

2020 ◽  
Vol 85 (3) ◽  
pp. 626-629
Author(s):  
Hisashi Muramatsu ◽  
Hiroki Maguchi ◽  
Taisuke Harada ◽  
Takehiro Kashiwagi ◽  
Chul-Sa Kim ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Propionic Acid ◽  
Unknown Function ◽  
Protein Family ◽  
Amino Acid Sequence Analysis ◽  
Gene Encoding

ABSTRACT Here, we report the identification of the gene encoding a novel enzyme, 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid desulfhydrase, in Burkholderia sp. HME13. The enzyme converts 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid and H2O to 3-(2,5-dioxoimidazolidin-4-yl) propionic acid and H2S. Amino acid sequence analysis of the enzyme indicates that it belongs to the DUF917 protein family, which consists of proteins of unknown function.

scholarly journals Catalytic and Molecular Properties of the Quinohemoprotein Tetrahydrofurfuryl Alcohol Dehydrogenase from Ralstonia eutropha Strain Bo

2001 ◽  
Vol 183 (6) ◽  
pp. 1954-1960 ◽  
Author(s):  
Grit Zarnt ◽  
Thomas Schräder ◽  
Jan R. Andreesen
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Alcohol Dehydrogenase ◽  
Tertiary Structure ◽  
Ralstonia Eutropha ◽  
Signal Sequence ◽  
Substrate Inhibition ◽  
Catalytic Efficiency ◽  
Gene Encoding

ABSTRACT The quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase (THFA-DH) from Ralstonia eutropha strain Bo was investigated for its catalytic properties. The apparentk cat/Km andK i values for several substrates were determined using ferricyanide as an artificial electron acceptor. The highest catalytic efficiency was obtained with n-pentanol exhibiting a k cat/Km value of 788 × 104 M−1 s−1. The enzyme showed substrate inhibition kinetics for most of the alcohols and aldehydes investigated. A stereoselective oxidation of chiral alcohols with a varying enantiomeric preference was observed. Initial rate studies using ethanol and acetaldehyde as substrates revealed that a ping-pong mechanism can be assumed for in vitro catalysis of THFA-DH. The gene encoding THFA-DH from R. eutropha strain Bo (tfaA) has been cloned and sequenced. The derived amino acid sequence showed an identity of up to 67% to the sequence of various quinoprotein and quinohemoprotein dehydrogenases. A comparison of the deduced sequence with the N-terminal amino acid sequence previously determined by Edman degradation analysis suggested the presence of a signal sequence of 27 residues. The primary structure of TfaA indicated that the protein has a tertiary structure quite similar to those of other quinoprotein dehydrogenases.

scholarly journals Characterization of Gmhsp26-A, a stress gene encoding a divergent heat shock protein of soybean: heavy-metal-induced inhibition of intron processing.

1988 ◽  
Vol 8 (3) ◽  
pp. 1113-1122 ◽  
Author(s):  
E Czarnecka ◽  
R T Nagao ◽  
J L Key ◽  
W B Gurley
Keyword(s):  
Amino Acid ◽  
Heat Shock ◽  
Amino Acid Sequence ◽  
Genomic Sequence ◽  
Stress Protein ◽  
Initiation Site ◽  
Gene Encoding ◽  
S1 Nuclease ◽  
Soybean Seedlings ◽  
Nuclease Mapping

We determined the DNA sequence and mapped the corresponding transcripts of a genomic clone containing the Gmhsp26-A gene of soybean. This gene is homologous to the previously characterized cDNA clone pCE54 (E. Czarnecka, L. Edelman, F. Schöffl, and J. L. Key, Plant Mol. Biol. 3:45-58, 1984) and is expressed in response to a wide variety of physiological stresses including heat shock (HS). S1 nuclease mapping of transcripts and a comparison of the cDNA sequence with the genomic sequence indicated the presence of a soybean seedlings with either CdCl2 or CuSO4. Analysis of the 5' termini of transcripts indicated the presence of one major and at least two minor start sites. In each case, initiation occurred 27 to 30 base pairs downstream from a TATA-like motif, and thus each initiation site appears to be promoted by the activity of a separate subpromoter. The three subpromoters are all associated with sequences showing low homology to the HS consensus element of Drosophila melanogaster HS genes and are differentially induced in response to various stresses. Within the carboxyl-terminal half of the protein, hydropathy analysis of the deduced amino acid sequence indicated a high degree of relatedness to the small HS proteins. A comparison of the primary amino acid sequence of hsp26-A with sequences of the small HS proteins suggested that this stress protein is highly diverged and may therefore be specialized for stress adaptation in soybean.

Characterization of Gmhsp26-A, a stress gene encoding a divergent heat shock protein of soybean: heavy-metal-induced inhibition of intron processing

1988 ◽  
Vol 8 (3) ◽  
pp. 1113-1122
Author(s):  
E Czarnecka ◽  
R T Nagao ◽  
J L Key ◽  
W B Gurley
Keyword(s):  
Amino Acid ◽  
Heat Shock ◽  
Amino Acid Sequence ◽  
Genomic Sequence ◽  
Stress Protein ◽  
Initiation Site ◽  
Gene Encoding ◽  
S1 Nuclease ◽  
Soybean Seedlings ◽  
Nuclease Mapping

We determined the DNA sequence and mapped the corresponding transcripts of a genomic clone containing the Gmhsp26-A gene of soybean. This gene is homologous to the previously characterized cDNA clone pCE54 (E. Czarnecka, L. Edelman, F. Schöffl, and J. L. Key, Plant Mol. Biol. 3:45-58, 1984) and is expressed in response to a wide variety of physiological stresses including heat shock (HS). S1 nuclease mapping of transcripts and a comparison of the cDNA sequence with the genomic sequence indicated the presence of a soybean seedlings with either CdCl2 or CuSO4. Analysis of the 5' termini of transcripts indicated the presence of one major and at least two minor start sites. In each case, initiation occurred 27 to 30 base pairs downstream from a TATA-like motif, and thus each initiation site appears to be promoted by the activity of a separate subpromoter. The three subpromoters are all associated with sequences showing low homology to the HS consensus element of Drosophila melanogaster HS genes and are differentially induced in response to various stresses. Within the carboxyl-terminal half of the protein, hydropathy analysis of the deduced amino acid sequence indicated a high degree of relatedness to the small HS proteins. A comparison of the primary amino acid sequence of hsp26-A with sequences of the small HS proteins suggested that this stress protein is highly diverged and may therefore be specialized for stress adaptation in soybean.

scholarly journals The EGD1 product, a yeast homolog of human BTF3, may be involved in GAL4 DNA binding.

1992 ◽  
Vol 12 (12) ◽  
pp. 5683-5689 ◽  
Author(s):  
M R Parthun ◽  
D A Mangus ◽  
J A Jaehning
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Amino Acid Sequence ◽  
Yeast Cells ◽  
Stable Complex ◽  
Affinity Column ◽  
Gene Encoding ◽  
Heat Stable ◽  
Gel Retardation ◽  
Final Purification Step

A variety of techniques, including filter binding, footprinting, and gel retardation, can be used to assay the transcriptional activator GAL4 (Gal4p) through the initial steps of its purification from yeast cells. Following DNA affinity chromatography, Gal4p still bound DNA selectively when assayed by filter binding or footprinting. However, the affinity-purified protein was no longer capable of forming a stable complex with DNA, as assayed by gel retardation. Mixing the purified Gal4p with the flowthrough fraction from the DNA affinity column restored gel retardation complex formation. Gel retardation assays were used to monitor the purification of a heat-stable Gal4p-DNA complex stabilization activity from the affinity column flowthrough. The activity coeluted from the final purification step with polypeptides of 21 and 27 kDa. The yeast gene encoding the 21-kDa protein was cloned on the basis of its N-terminal amino acid sequence. The gene, named EGD1 (enhancer of GAL4 DNA binding), encodes a highly basic protein (21% lysine and arginine) with a predicted molecular mass of 16.5 kDa. The amino acid sequence of the EGD1 product, Egd1p, is highly similar to that of the human protein BTF3 (X. M. Zheng, D. Black, P. Chambon, and J. M. Egly, Nature [London] 344:556-559, 1990). Although an egd1 null mutant was viable and Gal+, induction of the galactose-regulated genes in the egd1 mutant strain was significantly reduced when cells were shifted from glucose to galactose.

Pathogenesis-Related Proteins in Trifolium subterraneum: A General Survey and Subsequent Characterisation of a Protein Inducible by Ethephon and Redlegged Earth Mite Attack

1997 ◽  
Vol 24 (6) ◽  
pp. 819 ◽  
Author(s):  
Kevin Broderick ◽  
Christopher Pittock ◽  
Tony Arioli ◽  
Ernest H. Creaser ◽  
Jeremy J. Weinman ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Subterranean Clover ◽  
Trifolium Subterraneum ◽  
Chitinase Activity ◽  
Pr Proteins ◽  
Gene Encoding ◽  
Pathogenesis Related ◽  
Redlegged Earth Mite ◽  
Ethephon Treatment

One of the chief predators of subterranean clover (Trifolium subterraneum) pastures is redlegged earth mite (Halotydeus destructor; RLEM). Subterranean clover pathogenesis-related (PR) proteins induced by RLEM attack and ethephon treatment were surveyed, and PR proteins with peroxidase, β-1,3-glucanase and chitinase activities were detected. A protein co-migrating with a chitinase activity, induced by RLEM predation and treatment with ethephon, was isolated. It was purified and the N-terminal amino acid sequence determined. Using a degenerate oligonucleotide primer designed from this sequence, a corresponding cDNA fragment was amplified by reverse transcriptase-PCR, then cloned, and used as a probe to screen a subterranean clover cv. Karridale genomic library. The cDNA and a 97% homologous genomic clone were sequenced and the deduced amino acid sequence revealed an open reading frame of 157 amino acids capable of encoding a peptide of 16 478 Da. Significant homology (80%) was found between this protein and an abscisic acid (ABA)-responsive protein from Pisum sativum of unknown function which is an intracellular pathogenesis-related (IPR) protein. The gene encoding this protein also has homology to pea ‘disease response resistance genes’ and to proteins from other plant species in the PR-10 family. The induced protein was designated TsPR-10a due to its homology to other PR-10 proteins. Genomic Southern analysis indicates that the gene encoding this protein, designated Ypr10a, is a member of a multigene family with at least three members. Northern blot analysis indicates that the subterranean clover Ypr10a mRNA, or homologous transcript, level is strongly induced by ethephon treatment in both root and aerial tissues of 3 week old plants. The rapid induction kinetics of Ypr10a mRNA under ethephon treatment, its correlation with a putative chitinase activity, and homology to other PR-protein genes, suggests a pathogenesis-related role for TsPR-10a protein in subterranean clover.

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