scholarly journals Decarboxylating and Nondecarboxylating Glutaryl-Coenzyme A Dehydrogenases in the Aromatic Metabolism of Obligately Anaerobic Bacteria

2009 ◽  
Vol 191 (13) ◽  
pp. 4401-4409 ◽  
Author(s):  
Simon Wischgoll ◽  
Martin Taubert ◽  
Franziska Peters ◽  
Nico Jehmlich ◽  
Martin von Bergen ◽  
...  
Keyword(s):  
Coenzyme A ◽  
Anaerobic Bacteria ◽  
Oxidative Decarboxylation ◽  
Kinetic Properties ◽  
Strictly Anaerobic ◽  
Amino Acid Residues ◽  
Geobacter Metallireducens ◽  
Sulfate Reducing ◽  
Reverse Transcription Pcr ◽  
Gene Coding

ABSTRACT In anaerobic bacteria using aromatic growth substrates, glutaryl-coenzyme A (CoA) dehydrogenases (GDHs) are involved in the catabolism of the central intermediate benzoyl-CoA to three acetyl-CoAs and CO2. In this work, we studied GDHs from the strictly anaerobic, aromatic compound-degrading organisms Geobacter metallireducens (GDHGeo) (Fe[III] reducing) and Desulfococcus multivorans (GDHDes) (sulfate reducing). GDHGeo was purified from cells grown on benzoate and after the heterologous expression of the benzoate-induced bamM gene. The gene coding for GDHDes was identified after screening of a cosmid gene library. Reverse transcription-PCR revealed that its expression was induced by benzoate; the product was heterologously expressed and isolated. Both wild-type and recombinant GDHGeo catalyzed the oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA at similar rates. In contrast, recombinant GDHDes catalyzed only the dehydrogenation to glutaconyl-CoA. The latter compound was decarboxylated subsequently to crotonyl-CoA by the addition of membrane extracts from cells grown on benzoate in the presence of 20 mM NaCl. All GDH enzymes were purified as homotetramers of a 43- to 44-kDa subunit and contained 0.6 to 0.7 flavin adenine dinucleotides (FADs)/monomer. The kinetic properties for glutaryl-CoA conversion were as follows: for GDHGeo, the Km was 30 ± 2 μM and the V max was 3.2 ± 0.2 μmol min−1 mg−1, and for GDHDes, the Km was 52 ± 5 μM and the V max was 11 ± 1 μmol min−1 mg−1. GDHDes but not GDHGeo was inhibited by glutaconyl-CoA. Highly conserved amino acid residues that were proposed to be specifically involved in the decarboxylation of the intermediate glutaconyl-CoA were identified in GDHGeo but are missing in GDHDes. The differential use of energy-yielding/energy-demanding enzymatic processes in anaerobic bacteria that degrade aromatic compounds is discussed in view of phylogenetic relationships and constraints of overall energy metabolism.

scholarly journals Cyclohexa-1,5-Diene-1-Carbonyl-Coenzyme A (CoA) Hydratases of Geobacter metallireducens and Syntrophus aciditrophicus: Evidence for a Common Benzoyl-CoA Degradation Pathway in Facultative and Strict Anaerobes

2006 ◽  
Vol 189 (3) ◽  
pp. 1055-1060 ◽  
Author(s):  
Franziska Peters ◽  
Yoshifumi Shinoda ◽  
Michael J. McInerney ◽  
Matthias Boll
Keyword(s):  
Coenzyme A ◽  
Anaerobic Bacteria ◽  
Degradation Pathway ◽  
Ring Cleavage ◽  
Strictly Anaerobic ◽  
Significant Activity ◽  
Geobacter Metallireducens ◽  
Sulfate Reducing ◽  
Coa Reductase ◽  
Syntrophus Aciditrophicus

ABSTRACT In the denitrifying bacterium Thauera aromatica, the central intermediate of anaerobic aromatic metabolism, benzoyl-coenzyme A (CoA), is dearomatized by the ATP-dependent benzoyl-CoA reductase to cyclohexa-1,5-diene-1-carbonyl-CoA (dienoyl-CoA). The dienoyl-CoA is further metabolized by a series of β-oxidation-like reactions of the so-called benzoyl-CoA degradation pathway resulting in ring cleavage. Recently, evidence was obtained that obligately anaerobic bacteria that use aromatic growth substrates do not contain an ATP-dependent benzoyl-CoA reductase. In these bacteria, the reactions involved in dearomatization and cleavage of the aromatic ring have not been shown, so far. In this work, a characteristic enzymatic step of the benzoyl-CoA pathway in obligate anaerobes was demonstrated and characterized. Dienoyl-CoA hydratase activities were determined in extracts of Geobacter metallireducens (iron reducing), Syntrophus aciditrophicus (fermenting), and Desulfococcus multivorans (sulfate reducing) cells grown with benzoate. The benzoate-induced genes putatively coding for the dienoyl-CoA hydratases in the benzoate degraders G. metallireducens and S. aciditrophicus were heterologously expressed and characterized. Both gene products specifically catalyzed the reversible hydration of dienoyl-CoA to 6-hydroxycyclohexenoyl-CoA (Km , 80 and 35 μM; V max, 350 and 550 μmol min−1 mg−1, respectively). Neither enzyme had significant activity with cyclohex-1-ene-1-carbonyl-CoA or crotonyl-CoA. The results suggest that benzoyl-CoA degradation proceeds via dienoyl-CoA and 6-hydroxycyclohexanoyl-CoA in strictly anaerobic bacteria. The steps involved in dienoyl-CoA metabolism appear identical in all nonphotosynthetic anaerobic bacteria, although totally different benzene ring-dearomatizing enzymes are present in facultative and obligate anaerobes.

scholarly journals Purification and Characterization of Active-Site Components of the Putative p-Cresol Methylhydroxylase Membrane Complex from Geobacter metallireducens

2008 ◽  
Vol 190 (19) ◽  
pp. 6493-6500 ◽  
Author(s):  
Jörg Johannes ◽  
Alexander Bluschke ◽  
Nico Jehmlich ◽  
Martin von Bergen ◽  
Matthias Boll
Keyword(s):  
Competitive Inhibition ◽  
Anaerobic Bacteria ◽  
Functional Model ◽  
Visible Spectrum ◽  
Spectrometric Analysis ◽  
Strictly Anaerobic ◽  
Geobacter Metallireducens ◽  
Α Subunit ◽  
Hydroxybenzyl Alcohol

ABSTRACT p-Cresol methylhydroxylases (PCMH) from aerobic and facultatively anaerobic bacteria are soluble, periplasmic flavocytochromes that catalyze the first step in biological p-cresol degradation, the hydroxylation of the substrate with water. Recent results suggested that p-cresol degradation in the strictly anaerobic Geobacter metallireducens involves a tightly membrane-bound PCMH complex. In this work, the soluble components of this complex were purified and characterized. The data obtained suggest a molecular mass of 124 ± 15 kDa and a unique αα′β2 subunit composition, with α and α′ representing isoforms of the flavin adenine dinucleotide (FAD)-containing subunit and β representing a c-type cytochrome. Fluorescence and mass spectrometric analysis suggested that one FAD was covalently linked to Tyr394 of the α subunit. In contrast, the α′ subunit did not contain any FAD cofactor and is therefore considered to be catalytically inactive. The UV/visible spectrum was typical for a flavocytochrome with two heme c cofactors and one FAD cofactor. p-Cresol reduced the FAD but only one of the two heme cofactors. PCMH catalyzed both the hydroxylation of p-cresol to p-hydroxybenzyl alcohol and the subsequent oxidation of the latter to p-hydroxybenzaldehyde in the presence of artificial electron acceptors. The very low Km values (1.7 and 2.7 μM, respectively) suggest that the in vivo function of PCMH is to oxidize both p-cresol and p-hydroxybenzyl alcohol. The latter was a mixed inhibitor of p-cresol oxidation, with inhibition constants of a K ic (competitive inhibition) value of 18 ± 9 μM and a K iu (uncompetitive inhibition) value of 235 ± 20 μM. A putative functional model for an unusual PCMH enzyme is presented.

scholarly journals Structural and Functional Characterization of an Electron Transfer Flavoprotein Involved in Toluene Degradation in Strictly Anaerobic Bacteria

2019 ◽  
Vol 201 (21) ◽  
Author(s):  
Marian Samuel Vogt ◽  
Karola Schühle ◽  
Sebastian Kölzer ◽  
Patrick Peschke ◽  
Nilanjan Pal Chowdhury ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Conformational Changes ◽  
Functional Characterization ◽  
Anaerobic Bacteria ◽  
Biochemical Properties ◽  
Strictly Anaerobic ◽  
Toluene Degradation ◽  
Geobacter Metallireducens ◽  
Content Type ◽  
Facultatively Anaerobic

ABSTRACT (R)-Benzylsuccinate is the characteristic initial intermediate of anaerobic toluene metabolism, which is formed by a radical-type addition of toluene to fumarate. Its further degradation proceeds by activation to the coenzyme A (CoA)-thioester and β-oxidation involving a specific (R)-2-benzylsuccinyl-CoA dehydrogenase (BbsG) affiliated with the family of acyl-CoA dehydrogenases. In this report, we present the biochemical properties of electron transfer flavoproteins (ETFs) from the strictly anaerobic toluene-degrading species Geobacter metallireducens and Desulfobacula toluolica and the facultatively anaerobic bacterium Aromatoleum aromaticum. We determined the X-ray structure of the ETF paralogue involved in toluene metabolism of G. metallireducens, revealing strong overall similarities to previously characterized ETF variants but significantly different structural properties in the hinge regions mediating conformational changes. We also show that all strictly anaerobic toluene degraders utilize one of multiple genome-encoded related ETF paralogues, which constitute a distinct clade of similar sequences in the ETF family, for β-oxidation of benzylsuccinate. In contrast, facultatively anaerobic toluene degraders contain only one ETF species, which is utilized in all β-oxidation pathways. Our phylogenetic analysis of the known sequences of the ETF family suggests that at least 36 different clades can be differentiated, which are defined either by the taxonomic group of the respective host species (e.g., clade P for Proteobacteria) or by functional specialization (e.g., clade T for anaerobic toluene degradation). IMPORTANCE This study documents the involvement of ETF in anaerobic toluene metabolism as the physiological electron acceptor for benzylsuccinyl-CoA dehydrogenase. While toluene-degrading denitrifying proteobacteria use a common ETF species, which is also used for other β-oxidation pathways, obligately anaerobic sulfate- or ferric-iron-reducing bacteria use specialized ETF paralogues for toluene degradation. Based on the structure and sequence conservation of these ETFs, they form a new clade that is only remotely related to the previously characterized members of the ETF family. An exhaustive analysis of the available sequences indicated that the protein family consists of several closely related clades of proven or potential electron-bifurcating ETF species and many deeply branching nonbifurcating clades, which either follow the host phylogeny or are affiliated according to functional criteria.

scholarly journals Re-Citrate Synthase from Clostridium kluyveri Is Phylogenetically Related to Homocitrate Synthase and Isopropylmalate Synthase Rather Than to Si-Citrate Synthase

2007 ◽  
Vol 189 (11) ◽  
pp. 4299-4304 ◽  
Author(s):  
Fuli Li ◽  
Christoph H. Hagemeier ◽  
Henning Seedorf ◽  
Gerhard Gottschalk ◽  
Rudolf K. Thauer
Keyword(s):  
Primary Structure ◽  
Coenzyme A ◽  
Citrate Synthase ◽  
Anaerobic Bacteria ◽  
Strictly Anaerobic ◽  
Acetyl Coenzyme A ◽  
Isopropylmalate Synthase ◽  
Cell Extracts ◽  
Homocitrate Synthase ◽  
Clostridium Kluyveri

ABSTRACT The synthesis of citrate from acetyl-coenzyme A and oxaloacetate is catalyzed in most organisms by a Si-citrate synthase, which is Si-face stereospecific with respect to C-2 of oxaloacetate. However, in Clostridium kluyveri and some other strictly anaerobic bacteria, the reaction is catalyzed by a Re-citrate synthase, whose primary structure has remained elusive. We report here that Re-citrate synthase from C. kluyveri is the product of a gene predicted to encode isopropylmalate synthase. C. kluyveri is also shown to contain a gene for Si-citrate synthase, which explains why cell extracts of the organism always exhibit some Si-citrate synthase activity.

scholarly journals Structure and Function of the Unusual Tungsten Enzymes Acetylene Hydratase and Class II Benzoyl-Coenzyme A Reductase

2016 ◽  
Vol 26 (1-3) ◽  
pp. 119-137 ◽  
Author(s):  
Matthias Boll ◽  
Oliver Einsle ◽  
Ulrich Ermler ◽  
Peter M.H. Kroneck ◽  
G. Matthias Ullmann
Keyword(s):  
Alkali Metals ◽  
Coenzyme A ◽  
Anaerobic Bacteria ◽  
Class Ii ◽  
Structure And Function ◽  
Organic Chemical ◽  
Strictly Anaerobic ◽  
Dimethyl Sulfoxide Reductase ◽  
Coa Reductase ◽  
And Function

In biology, tungsten (W) is exclusively found in microbial enzymes bound to a bis<i>-</i>pyranopterin cofactor (bis-WPT). Previously known W enzymes catalyze redox oxo/hydroxyl transfer reactions by directly coordinating their substrates or products to the metal. They comprise the W-containing formate/formylmethanofuran dehydrogenases belonging to the dimethyl sulfoxide reductase (DMSOR) family and the aldehyde:ferredoxin oxidoreductase (AOR) families, which form a separate enzyme family within the Mo/W enzymes. In the last decade, initial insights into the structure and function of two unprecedented W enzymes were obtained: the acetaldehyde forming acetylene hydratase (ACH) belongs to the DMSOR and the class II benzoyl-coenzyme A (CoA) reductase (BCR) to the AOR family. The latter catalyzes the reductive dearomatization of benzoyl-CoA to a cyclic diene. Both are key enzymes in the degradation of acetylene (ACH) or aromatic compounds (BCR) in strictly anaerobic bacteria. They are unusual in either catalyzing a nonredox reaction (ACH) or a redox reaction without coordinating the substrate or product to the metal (BCR). In organic chemical synthesis, analogous reactions require totally nonphysiological conditions depending on Hg<sup>2+</sup> (acetylene hydration) or alkali metals (benzene ring reduction). The structural insights obtained pave the way for biological or biomimetic approaches to basic reactions in organic chemistry.

Pilot Plant Study on Microaerobic Self-Granulated Sludge Process (Multi-Stage Reversing Flow Bioreactor: MRB)

1991 ◽  
Vol 23 (4-6) ◽  
pp. 973-980 ◽  
Author(s):  
M. Takahashi ◽  
S. Kyosai
Keyword(s):  
Pilot Plant ◽  
High Performance ◽  
Anaerobic Bacteria ◽  
Domestic Wastewater ◽  
Public Works ◽  
Symbiotic Interaction ◽  
Sulfate Reducing ◽  
Pellet Formation ◽  
Multi Stage ◽  
Domestic Wastewater Treatment

A Multi-stage Reversing flow Bioreactor (MRB) was developed by the Public Works Research Institute in 1986. It utilizes the symbiotic interaction between anaerobic bacteria (sulfate reducing bacteria) and microaerobic bacteria (Beggiatoa=filamentous sulfur oxidizing bacteria) for self-granulated pellet formation. A MRB Pilot plant for domestic wastewater treatment (design capacity was 225 m3/day) was constructed in 1988. After several modifications of the initial design, stable pellet formation and high performance were achieved. This paper describes the results of the pilot plant operation.

scholarly journals ABCA5 Resides in Lysosomes, and ABCA5 Knockout Mice Develop Lysosomal Disease-Like Symptoms

2005 ◽  
Vol 25 (10) ◽  
pp. 4138-4149 ◽  
Author(s):  
Yoshiyuki Kubo ◽  
Sayaka Sekiya ◽  
Megumi Ohigashi ◽  
Chiemi Takenaka ◽  
Kyoko Tamura ◽  
...  
Keyword(s):  
Thyroid Gland ◽  
Abc Transporter ◽  
Neural Cells ◽  
Newborn Mouse ◽  
Amino Acid Residues ◽  
Lysosomal Disease ◽  
Transmembrane Segments ◽  
Reverse Transcription Pcr ◽  
Localization Analysis ◽  
Immunohistochemical Studies

ABSTRACT ABCA5 is a member of the ABC transporter A subfamily, and a mouse orthologue (mABCA5) in newborn mouse brain and neural cells was identified by reverse transcription-PCR. Full-length cDNA cloning revealed that mABCA5 consists of 1,642 amino acid residues and that its putative structure is that of a full-type ABC transporter having two sets of six transmembrane segments and a nucleotide binding domain. Immunohistochemical studies revealed that mABCA5 is expressed in brain, lung, heart, and thyroid gland. A subcellular localization analysis showed that mABCA5 is a resident of lysosomes and late endosomes. Abca5 − / − mice exhibited symptoms similar to those of several lysosomal diseases in heart, although no prominent abnormalities were found in brain or lung. They developed a dilated cardiomyopathy-like heart after reaching adulthood and died due to depression of the cardiovascular system. In addition, Abca5 − / − mice also exhibited exophthalmos and collapse of the thyroid gland. Therefore, ABCA5 is a protein related to a lysosomal disease and plays important roles, especially in cardiomyocytes and follicular cells.

scholarly journals Ribose 5-phosphate isomerase type B from Trypanosoma cruzi: kinetic properties and site-directed mutagenesis reveal information about the reaction mechanism

2006 ◽  
Vol 401 (1) ◽  
pp. 279-285 ◽  
Author(s):  
Ana L. Stern ◽  
Emmanuel Burgos ◽  
Laurent Salmon ◽  
Juan J. Cazzulo
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Pentose Phosphate ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Type B ◽  
Nucleotide Synthesis ◽  
Gene Coding ◽  
Genes Encoding

Trypanosoma cruzi, the human parasite that causes Chagas disease, contains a functional pentose phosphate pathway, probably essential for protection against oxidative stress and also for R5P (ribose 5-phosphate) production for nucleotide synthesis. The haploid genome of the CL Brener clone of the parasite contains one gene coding for a Type B Rpi (ribose 5-phosphate isomerase), but genes encoding Type A Rpis, most frequent in eukaryotes, seem to be absent. The RpiB enzyme was expressed in Escherichia coli as a poly-His tagged active dimeric protein, which catalyses the reversible isomerization of R5P to Ru5P (ribulose 5-phos-phate) with Km values of 4 mM (R5P) and 1.4 mM (Ru5P).4-Phospho-D-erythronohydroxamic acid, an analogue to the reaction intermediate when the Rpi acts via a mechanism involving the formation of a 1,2-cis-enediol, inhibited the enzyme competi-tively, with an IC50 value of 0.7 mM and a Ki of 1.2 mM. Site-directed mutagenesis allowed the demonstration of a role for His102, but not for His138, in the opening of the ribose furanosic ring. A major role in catalysis was confirmed for Cys69, since the C69A mutant was inactive in both forward and reverse directions of the reaction. The present paper contributes to the know-ledge of the mechanism of the Rpi reaction; in addition, the absence of RpiBs in the genomes of higher animals makes this enzyme a possible target for chemotherapy of Chagas disease.

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