scholarly journals Genes Coding for a New Pathway of Aerobic Benzoate Metabolism in Azoarcus evansii

2002 ◽  
Vol 184 (22) ◽  
pp. 6301-6315 ◽  
Author(s):  
Johannes Gescher ◽  
Annette Zaar ◽  
Magdy Mohamed ◽  
Hermann Schägger ◽  
Georg Fuchs
Keyword(s):  
Bacillus Stearothermophilus ◽  
Organic Substrate ◽  
Amino Acid Sequences ◽  
Ring Cleavage ◽  
Shikimate Kinase ◽  
Coa Ligase ◽  
Regulator Protein ◽  
New Protein Families ◽  
New Type ◽  
Coa Thioesters

ABSTRACT A new pathway for aerobic benzoate oxidation has been postulated for Azoarcus evansii and for a Bacillus stearothermophilus-like strain. Benzoate is first transformed into benzoyl coenzyme A (benzoyl-CoA), which subsequently is oxidized to 3-hydroxyadipyl-CoA and then to 3-ketoadipyl-CoA; all intermediates are CoA thioesters. The genes coding for this benzoate-induced pathway were investigated in the β-proteobacterium A. evansii. They were identified on the basis of N-terminal amino acid sequences of purified benzoate metabolic enzymes and of benzoate-induced proteins identified on two-dimensional gels. Fifteen genes probably coding for the benzoate pathway were found to be clustered on the chromosome. These genes code for the following functions: a putative ATP-dependent benzoate transport system, benzoate-CoA ligase, a putative benzoyl-CoA oxygenase, a putative isomerizing enzyme, a putative ring-opening enzyme, enzymes for β-oxidation of CoA-activated intermediates, thioesterase, and lactone hydrolase, as well as completely unknown enzymes belonging to new protein families. An unusual putative regulator protein consists of a regulator protein and a shikimate kinase I-type domain. A deletion mutant with a deletion in one gene (boxA) was unable to grow with benzoate as the sole organic substrate, but it was able to grow with 3-hydroxybenzoate and adipate. The data support the proposed pathway, which postulates operation of a new type of ring-hydroxylating dioxygenase acting on benzoyl-CoA and nonoxygenolytic ring cleavage. A β-oxidation-like metabolism of the ring cleavage product is thought to lead to 3-ketoadipyl-CoA, which finally is cleaved into succinyl-CoA and acetyl-CoA.

scholarly journals Two Similar Gene Clusters Coding for Enzymes of a New Type of Aerobic 2-Aminobenzoate (Anthranilate) Metabolism in the BacteriumAzoarcus evansii

2001 ◽  
Vol 183 (18) ◽  
pp. 5268-5278 ◽  
Author(s):  
Karola Schühle ◽  
Martina Jahn ◽  
Sandro Ghisla ◽  
Georg Fuchs
Keyword(s):  
Gene Clusters ◽  
Old Yellow Enzyme ◽  
Coding Regions ◽  
System A ◽  
Coa Ligase ◽  
Genes Encoding ◽  
Intergenic Regions ◽  
New Type ◽  
Coa Thioesters ◽  
Substrate Binding Protein

ABSTRACT In the β-proteobacterium Azoarcus evansii, the aerobic metabolism of 2-aminobenzoate (anthranilate), phenylacetate, and benzoate proceeds via three unprecedented pathways. The pathways have in common that all three substrates are initially activated to coenzyme A (CoA) thioesters and further processed in this form. The two initial steps of 2-aminobenzoate metabolism are catalyzed by a 2-aminobenzoate-CoA ligase forming 2-aminobenzoyl-CoA and by a 2-aminobenzoyl-CoA monooxygenase/reductase (ACMR) forming 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA. Eight genes possibly involved in this pathway, including the genes encoding 2-aminobenzoate-CoA ligase and ACMR, were detected, cloned, and sequenced. The sequence of the ACMR gene showed that this enzyme is an 87-kDa fusion protein of two flavoproteins, a monooxygenase (similar to salicylate monooxygenase) and a reductase (similar to old yellow enzyme). Besides the genes for the initial two enzymes, genes for three enzymes of a β-oxidation pathway were found. A substrate binding protein of an ABC transport system, a MarR-like regulator, and a putative translation inhibitor protein were also encoded by the gene cluster. The data suggest that, after monooxygenation/reduction of 2-aminobenzoyl-CoA, the nonaromatic CoA thioester intermediate is metabolized further by β-oxidation. This implies that all subsequent intermediates are CoA thioesters and that the alicyclic carbon ring is not cleaved oxygenolytically. Surprisingly, the cluster of eight genes, which form an operon, is duplicated. The two copies differ only marginally within the coding regions but differ substantially in the respective intergenic regions. Both copies of the genes are coordinately expressed in cells grown aerobically on 2-aminobenzoate.

scholarly journals Characterization of the Gallate Dioxygenase Gene: Three Distinct Ring Cleavage Dioxygenases Are Involved in Syringate Degradation by Sphingomonas paucimobilis SYK-6

2005 ◽  
Vol 187 (15) ◽  
pp. 5067-5074 ◽  
Author(s):  
Daisuke Kasai ◽  
Eiji Masai ◽  
Keisuke Miyauchi ◽  
Yoshihiro Katayama ◽  
Masao Fukuda
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Terminal Region ◽  
Ring Cleavage ◽  
Sphingomonas Paucimobilis ◽  
Acid Protein ◽  
Demethylation Pathway ◽  
Dioxygenase Gene ◽  
Amino Acid Protein

ABSTRACT Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate (PCA) and 3-O-methylgallate (3MGA) in reactions with the tetrahydrofolate-dependent O-demethylases LigM and DesA, respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, whereas 3MGA is metabolized via three distinct pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and 3MGA O-demethylase (LigM) are involved. In the 3MGA O-demethylation pathway, LigM converts 3MGA to gallate, and the resulting gallate appears to be degraded by a dioxygenase other than LigAB or DesZ. Here, we isolated the gallate dioxygenase gene, desB, which encodes a 418-amino-acid protein with a molecular mass of 46,843 Da. The amino acid sequences of the N-terminal region (residues 1 to 285) and the C-terminal region (residues 286 to 418) of DesB exhibited ca. 40% and 27% identity with the sequences of the PCA 4,5-dioxygenase β and α subunits, respectively. DesB produced in Escherichia coli was purified and was estimated to be a homodimer (86 kDa). DesB specifically attacked gallate to generate 4-oxalomesaconate as the reaction product. The Km for gallate and the V max were determined to be 66.9 ± 9.3 μM and 42.7 ± 2.4 U/mg, respectively. On the basis of the analysis of various SYK-6 mutants lacking the genes involved in syringate degradation, we concluded that (i) all of the three-ring cleavage dioxygenases are involved in syringate catabolism, (ii) the pathway involving LigM and DesB plays an especially important role in the growth of SYK-6 on syringate, and (iii) DesB and LigAB are involved in gallate degradation.

scholarly journals Epoxy Coenzyme A Thioester Pathways for Degradation of Aromatic Compounds

2012 ◽  
Vol 78 (15) ◽  
pp. 5043-5051 ◽  
Author(s):  
Wael Ismail ◽  
Johannes Gescher
Keyword(s):  
Aromatic Ring ◽  
Aromatic Compounds ◽  
Coenzyme A ◽  
Environmental Pollutants ◽  
Bacterial Degradation ◽  
Ring Cleavage ◽  
The Novel ◽  
Energy Consuming ◽  
Coa Thioesters ◽  
Hydrolytic Mechanism

ABSTRACTAromatic compounds (biogenic and anthropogenic) are abundant in the biosphere. Some of them are well-known environmental pollutants. Although the aromatic nucleus is relatively recalcitrant, microorganisms have developed various catabolic routes that enable complete biodegradation of aromatic compounds. The adopted degradation pathways depend on the availability of oxygen. Under oxic conditions, microorganisms utilize oxygen as a cosubstrate to activate and cleave the aromatic ring. In contrast, under anoxic conditions, the aromatic compounds are transformed to coenzyme A (CoA) thioesters followed by energy-consuming reduction of the ring. Eventually, the dearomatized ring is opened via a hydrolytic mechanism. Recently, novel catabolic pathways for the aerobic degradation of aromatic compounds were elucidated that differ significantly from the established catabolic routes. The new pathways were investigated in detail for the aerobic bacterial degradation of benzoate and phenylacetate. In both cases, the pathway is initiated by transforming the substrate to a CoA thioester and all the intermediates are bound by CoA. The subsequent reactions involve epoxidation of the aromatic ring followed by hydrolytic ring cleavage. Here we discuss the novel pathways, with a particular focus on their unique features and occurrence as well as ecological significance.

scholarly journals Characterization of the Genes for Two Protocatechuate 3,4-Dioxygenases from the 4-Sulfocatechol-Degrading Bacterium Agrobacterium radiobacter Strain S2

2000 ◽  
Vol 182 (21) ◽  
pp. 6123-6129 ◽  
Author(s):  
Matthias Contzen ◽  
Andreas Stolz
Keyword(s):  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Agrobacterium Radiobacter ◽  
Sequence Alignments ◽  
Degrading Bacterium ◽  
Regulator Protein ◽  
Putative Operon ◽  
Genes Encoding ◽  
Reading Frames

ABSTRACT The genes for two different protocatechuate 3,4-dioxygenases (P34Os) were cloned from the 4-sulfocatechol-degrading bacteriumAgrobacterium radiobacter strain S2 (DSMZ 5681). ThepcaH1G1 genes encoded a P34O (P34O-I) which oxidized protocatechuate but not 4-sulfocatechol. These genes were part of a protocatechuate-degradative operon which strongly resembled the isofunctional operon from the protocatechuate-degrading strainAgrobacterium tumefaciens A348 described previously by D. Parke (FEMS Microbiol. Lett. 146:3–12, 1997). The second P34O (P34O-II), encoded by the pcaH2G2 genes, was functionally expressed and shown to convert protocatechuate and 4-sulfocatechol. A comparison of the deduced amino acid sequences of PcaH-I and PcaH-II, and of PcaG-I and PcaG-II, with each other and with the corresponding sequences from the P34Os, from other bacterial genera suggested that the genes for the P34O-II were obtained by strain S2 by lateral gene transfer. The genes encoding the P34O-II were found in a putative operon together with two genes which, according to sequence alignments, encoded transport proteins. Further downstream from this putative operon, two open reading frames which code for a putative regulator protein of the IclR family and a putative 3-carboxymuconate cycloisomerase were identified.

scholarly journals The bzd Gene Cluster, Coding for Anaerobic Benzoate Catabolism, in Azoarcus sp. Strain CIB

2004 ◽  
Vol 186 (17) ◽  
pp. 5762-5774 ◽  
Author(s):  
María J. López Barragán ◽  
Manuel Carmona ◽  
María T. Zamarro ◽  
Bärbel Thiele ◽  
Matthias Boll ◽  
...  
Keyword(s):  
Degradation Pathway ◽  
Ring Cleavage ◽  
Gene Products ◽  
Physiological Control ◽  
Significant Similarity ◽  
Thauera Aromatica ◽  
Benzoate Degradation ◽  
Coa Ligase ◽  
Transcriptional Units ◽  
Coa Reductase

ABSTRACT We report here that the bzd genes for anaerobic benzoate degradation in Azoarcus sp. strain CIB are organized as two transcriptional units, i.e., a benzoate-inducible catabolic operon, bzdNOPQMSTUVWXYZA, and a gene, bzdR, encoding a putative transcriptional regulator. The last gene of the catabolic operon, bzdA, has been expressed in Escherichia coli and encodes the benzoate-coenzyme A (CoA) ligase that catalyzes the first step in the benzoate degradation pathway. The BzdA enzyme is able to activate a wider range of aromatic compounds than that reported for other previously characterized benzoate-CoA ligases. The reduction of benzoyl-CoA to a nonaromatic cyclic intermediate is carried out by a benzoyl-CoA reductase (bzdNOPQ gene products) detected in Azoarcus sp. strain CIB extracts. The bzdW, bzdX, and bzdY gene products show significant similarity to the hydratase, dehydrogenase, and ring-cleavage hydrolase that act sequentially on the product of the benzoyl-CoA reductase in the benzoate catabolic pathway of Thauera aromatica. Benzoate-CoA ligase assays and transcriptional analyses based on lacZ-reporter fusions revealed that benzoate degradation in Azoarcus sp. strain CIB is subject to carbon catabolite repression by some organic acids, indicating the existence of a physiological control that connects the expression of the bzd genes to the metabolic status of the cell.

scholarly journals Cloning and Sequencing of a Novel meta-Cleavage Dioxygenase Gene Whose Product Is Involved in Degradation of γ-Hexachlorocyclohexane in Sphingomonas paucimobilis

1999 ◽  
Vol 181 (21) ◽  
pp. 6712-6719 ◽  
Author(s):  
Keisuke Miyauchi ◽  
Yugo Adachi ◽  
Yuji Nagata ◽  
Masamichi Takagi
Keyword(s):  
Amino Acids ◽  
Sole Source ◽  
Ring Cleavage ◽  
Hydroxyl Groups ◽  
Aromatic Rings ◽  
Cloning And Sequencing ◽  
Direct Demonstration ◽  
Rate Of Reaction ◽  
Dioxygenase Gene ◽  
New Type

ABSTRACT Sphingomonas (formerly Pseudomonas)paucimobilis UT26 utilizes γ-hexachlorocyclohexane (γ-HCH), a halogenated organic insecticide, as a sole source of carbon and energy. In a previous study, we showed that γ-HCH is degraded to chlorohydroquinone (CHQ) and then to hydroquinone (HQ), although the rate of reaction from CHQ to HQ was slow (K. Miyauchi, S. K. Suh, Y. Nagata, and M. Takagi, J. Bacteriol. 180:1354–1359, 1998). In this study, we cloned and characterized a gene, designated linE, which is located upstream oflinD and is directly involved in the degradation of CHQ. The LinE protein consists of 321 amino acids, and all of the amino acids which are reported to be essential for the activity ofmeta-cleavage dioxygenases are conserved in LinE.Escherichia coli overproducing LinE could convert both CHQ and HQ, producing γ-hydroxymuconic semialdehyde and maleylacetate, respectively, with consumption of O2 but could not convert catechol, which is one of the major substrates formeta-cleavage dioxygenases. LinE seems to be resistant to the acylchloride, which is the ring cleavage product of CHQ and which seems to react with water to be converted to maleylacetate. These results indicated that LinE is a novel type ofmeta-cleavage dioxygenase, designated (chloro)hydroquinone 1,2-dioxygenase, which cleaves aromatic rings with two hydroxyl groups at para positions preferably. This study represents a direct demonstration of a new type of ring cleavage pathway for aromatic compounds, the hydroquinone pathway.

scholarly journals The Biosynthetic Pathway of Major Avenanthramides in Oat

Metabolites ◽  
2019 ◽  
Vol 9 (8) ◽  
pp. 163 ◽  
Author(s):  
Zhiyong Li ◽  
Yi Chen ◽  
Dauenpen Meesapyodsuk ◽  
Xiao Qiu
Keyword(s):  
Biosynthetic Pathway ◽  
Molecular Mechanisms ◽  
Functional Characterization ◽  
Acyl Donor ◽  
Acyl Acceptor ◽  
Coa Ligase ◽  
Health Promoting ◽  
Hydroxyanthranilic Acid ◽  
Coa Thioesters

Avenanthramides are a group of N-cinnamoylanthranilic acids, with health-promoting properties mainly found in oat (Avena sativa L.). However, the biosynthetic mechanism for the main three types of avenanthramides (Avn-A, Avn-B and Avn-C) is not completely understood. In the present study, we report molecular identification and functional characterization of three different types of genes from oat encoding 4-coumarate-CoA ligase (4CL), hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT) and a caffeoyl-CoA O-methyltransferase (CCoAOMT) enzymes, all involved in the biosynthesis of these avenanthramides. In vitro enzymatic assays using the proteins expressed in Escherichia coli showed that oat 4CL could convert p-coumaric acid, caffeic acid and ferulic acid to their CoA thioesters. Oat HHTs were only responsible for the biosynthesis of Avn-A and Avn-C using hydroxyanthranilic acid as an acyl acceptor and p-coumaroyl-CoA and caffeoyl-CoA as an acyl donor, respectively. Avn-B was synthesized by a CCoAOMT enzyme through the methylation of Avn-C. Collectively, these results have elucidated the molecular mechanisms for the biosynthesis of three major avenanthramides in vitro and paved the way for metabolic engineering of the biosynthetic pathway in heterologous systems to produce nutraceutically important compounds and make possible genetic improvement of this nutritional trait in oat through marker-assisted breeding.

Is The SARS-CoV2 evolved in Human Being: A prospective Genetic Analysis

2020 ◽  
pp. 169-177
Author(s):  
Salvatore Dimonte ◽  
Paywast Jamal Jalal ◽  
Taib Ahmed Hama Soor ◽  
Safa Bakr Karim ◽  
Hiwa Abdulrahman Ahmad ◽  
...  
Keyword(s):  
Phylogenetic Trees ◽  
Rna Binding ◽  
Unknown Origin ◽  
Amino Acid Sequences ◽  
Recent Effort ◽  
Human Coronavirus ◽  
Evolutionary Changes ◽  
New Type ◽  
Human Coronaviruses ◽  
Novel Coronavirus

COVID-19 is the deadly respiratory disease of the century caused by new type unknown origin Coronavirus. The recent effort of the word researchers is toward finding the origin of the virus. The current study investigated the extent of molecular similarity and divergence between SARS-CoV2 and other related Coronavirus. An attempt has been made to investigate the epidemiological study of this new contagious virus using molecular biology techniques. The phylogenetic trees for all human coronaviruses with the novel Coronavirus have been built using a several complete amino acid sequences of the four known structural proteins, S (spike), E (envelope), M (membrane), and N (nucleocapsid). The result of the study revealed that the SARS-CoV2 is related to human SARS-CoV isolated from different countries very cloely, especially those strains recovered from China in recent times, 2020. The evolutionary changes observed in the inserted 23 amino acids in the RNA binding domain (RBD) of the coronvirus spike glycoprotein which cannot be detected in any other human coronavirus. Moreover, the 2019-nCoV is not closely related to other alpha, beta and gamma human Coronavirus, including MERS-CoV. The current study concluded that 2019-nCoV is more likely believed to originated from SARS-CoV. The probability is more vital to be originated from the strain isolated in China in 2020, which is coincident with the spraed of COVID-19 in the same country. The phyloepidemiologic analyses suggested that the coronaviruses are circulating in human hosts evolving gradually by times in response to the different environment stimuli facing the virus inside the host in different geographical areas. Furthermore, the analysis showed the flow of transmission, and evolutionary changes of SARS-CoV2 which may be directed from the transmission of SARS-CoV from human to Bat and Pangolin then jumped to human again in the crowded market Wuhan city in China.

scholarly journals Characterization of the 4-Hydroxybenzoyl-Coenzyme A Thioesterase from Arthrobacter sp. Strain SU

2003 ◽  
Vol 69 (5) ◽  
pp. 2707-2711 ◽  
Author(s):  
Zhihao Zhuang ◽  
Karl-Heinz Gartemann ◽  
Rudolf Eichenlaub ◽  
Debra Dunaway-Mariano
Keyword(s):  
Coenzyme A ◽  
Sequence Data ◽  
Protein A ◽  
Coa Ligase ◽  
Protein Sequence Data ◽  
Coa Thioesters ◽  
Ph Range ◽  
Rate Profile ◽  
Encoding Genes

ABSTRACT The Arthrobacter sp. strain SU 4-chlorobenzoate (4-CBA) dehalogenation pathway converts 4-CBA to 4-hydroxybenzoate (4-HBA). The pathway operon contains the genes fcbA, fcbB, and fcbC (A. Schmitz, K. H. Gartemann, J. Fiedler, E. Grund, and R. Eichenlaub, Appl. Environ. Microbiol. 58:4068-4071, 1992). Genes fcbA and fcbB encode 4-CBA-coenzyme A (CoA) ligase and 4-CBA-CoA dehalogenase, respectively, whereas the function of fcbC is not known. We subcloned fcbC and expressed it in Escherichia coli, and we purified and characterized the FcbC protein. A substrate activity screen identified benzoyl-CoA thioesters as the most active substrates. Catalysis of 4-HBA-CoA hydrolysis to 4-HBA and CoA occurred with a k cat of 6.7 s−1 and a Km of 1.2 μM. The k cat pH rate profile for 4-HBA-CoA hydrolysis indicated optimal activity over a pH range of 6 to 10. The amino acid sequence of the FcbC protein was compared to other sequences contained in the protein sequence data banks. A large number of sequence homologues of unknown function were identified. On the other hand, the 4-HBA-CoA thioesterases isolated from 4-CBA-degrading Pseudomonas strains did not share significant sequence identity with the FcbC protein, indicating early divergence of the thioesterase-encoding genes.

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