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 Genes, Enzymes, and Regulation of para-Cresol Metabolism in Geobacter metallireducens

2007 ◽  
Vol 189 (13) ◽  
pp. 4729-4738 ◽  
Author(s):  
Franziska Peters ◽  
Dimitri Heintz ◽  
Jörg Johannes ◽  
Alain van Dorsselaer ◽  
Matthias Boll
Keyword(s):  
Alcohol Oxidation ◽  
Regulatory Elements ◽  
Denitrifying Bacteria ◽  
Regulation Of Transcription ◽  
Strictly Anaerobic ◽  
Geobacter Metallireducens ◽  
Α Subunit ◽  
Proteomic Approach ◽  
Activity Measurements ◽  
Hydroxybenzyl Alcohol

ABSTRACT In aerobic and facultatively anaerobic bacteria, the degradation of para-cresol (p-cresol) involves the initial hydroxylation to p-hydroxybenzyl alcohol by water catalyzed by the soluble, periplasmatic flavocytochrome p-cresol methylhydroxylase (PCMH; α2β2 composition). In denitrifying bacteria the further metabolism proceeds via oxidation to p-hydroxybenzoate, the formation of p-hydroxybenzoyl-coenzyme A (CoA), and the subsequent dehydroxylation of the latter to benzoyl-CoA by reduction. In contrast, the strictly anaerobic Desulfobacterium cetonicum degrades p-cresol by addition to fumarate, yielding p-hydroxybenzylsuccinate. In this work, in vitro enzyme activity measurements revealed that the obligately anaerobic Geobacter metallireducens uses the p-cresol degradation pathway of denitrifying bacteria. Surprisingly, PCMH, which is supposed to catalyze both p-cresol hydroxylation and p-hydroxybenzyl alcohol oxidation to the corresponding aldehyde, was located in the membrane fraction. The α subunit of the enzyme was present in two isoforms, suggesting an αα′β2 composition. We propose that the unusual asymmetric architecture and the membrane association of PCMH might be important for alternative electron transfer routes to either cytochrome c (in the case of p-cresol oxidation) or to menaquinone (in the case of p-hydroxybenzyl alcohol oxidation). Unusual properties of further enzymes of p-cresol metabolism, p-hydroxybenzoate-CoA ligase, and p-hydroxybenzoyl-CoA reductase were identified and are discussed. A proteomic approach identified a gene cluster comprising most of the putative structural genes for enzymes involved in p-cresol metabolism (pcm genes). Reverse transcription-PCR studies revealed a different regulation of transcription of pcm genes and the corresponding enzyme activities, suggesting the presence of posttranscriptional regulatory elements.

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 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 Kinetic and regulatory properties of cytosolic pyruvate kinase from germinating castor oil seeds

1991 ◽  
Vol 279 (2) ◽  
pp. 495-501 ◽  
Author(s):  
F E Podestá ◽  
W C Plaxton
Keyword(s):  
Pyruvate Kinase ◽  
Castor Oil ◽  
Mixed Type ◽  
Competitive Inhibition ◽  
Ricinus Communis ◽  
Cytosolic Ph ◽  
Oil Seeds ◽  
Saturation Kinetics ◽  
Regulatory Properties

The kinetic and regulatory properties of cytosolic pyruvate kinase (PKc) isolated from endosperm of germinating castor oil seeds (Ricinus communis L.) have been studied. Optimal efficiency in substrate utilization (in terms of Vmax/Km for phosphoenolpyruvate or ADP) occurred between pH 6.7 and 7.4. Enzyme activity was absolutely dependent on the presence of a bivalent and a univalent metal cation, with Mg2+ and K+ fulfilling this requirement. Mg2+ binding showed positive and negative co-operativity at pH 6.5 (h = 1.6) and pH 7.2 (h = 0.69) respectively. Hyperbolic saturation kinetics were observed with phosphoenolpyruvate (PEP) and K+, whereas ADP acted as a mixed-type inhibitor over 1 mM. Glycerol (10%, v/v) increased the S0.5(ADP) 2.3-fold and altered the pattern of nucleotide binding from hyperbolic (h = 1.0) to sigmoidal (h = 1.79) without modifying PEP saturation kinetics. No activators were identified. ATP, AMP, isocitrate, 2-oxoglutarate, malate, 2-phosphoglycerate, 2,3-bisphosphoglycerate, 3-phosphoglycerate, glycerol 3-phosphate and phosphoglycolate were the most effective inhibitors. These metabolites yielded additive inhibition when tested in pairs. ATP and 3-phosphoglycerate were mixed-type inhibitors with respect to PEP, whereas competitive inhibition was observed for other inhibitors. Inhibition by malate, 2-oxoglutarate, phosphorylated triose sugars or phosphoglycolate was far more pronounced at pH 7.2 than at pH 6.5. Although 32P-labelling studies revealed that extensive phosphorylation in vivo of soluble endosperm proteins occurred between days 3 and 5 of seed germination, no alteration in the 32P-labelling pattern of 5-day-germinated endosperm was observed after 30 min of anaerobiosis. Moreover, no evidence was obtained that PKc was a phosphoprotein in aerobic or anoxic endosperms. It is proposed that endosperm PKc activity of germinating castor seeds is enhanced after anaerobiosis through concerted decreases in ATP levels, cytosolic pH and concentrations of several key inhibitors.

scholarly journals Hsp90 Binds and Regulates the Ligand-Inducible α Subunit of Eukaryotic Translation Initiation Factor Kinase Gcn2

1999 ◽  
Vol 19 (12) ◽  
pp. 8422-8432 ◽  
Author(s):  
Olivier Donzé ◽  
Didier Picard
Keyword(s):  
Amino Acid ◽  
Constitutive Expression ◽  
Amino Acid Starvation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Restrictive Temperature ◽  
Α Subunit ◽  
Macbecin I

ABSTRACT The protein kinase Gcn2 stimulates translation of the yeast transcription factor Gcn4 upon amino acid starvation. Using genetic and biochemical approaches, we show that Gcn2 is regulated by the molecular chaperone Hsp90 in budding yeast Saccharomyces cerevisiae. Specifically, we found that (i) several Hsp90 mutant strains exhibit constitutive expression of a GCN4-lacZ reporter plasmid; (ii) Gcn2 and Hsp90 form a complex in vitro as well as in vivo; (iii) the specific inhibitors of Hsp90, geldanamycin and macbecin I, enhance the association of Gcn2 with Hsp90 and inhibit its kinase activity in vitro; (iv) in vivo, macbecin I strongly reduces the levels of Gcn2; (v) in a strain expressing the temperature-sensitive Hsp90 mutant G170D, both the accumulation and activity of Gcn2 are abolished at the restrictive temperature; and (vi) the Hsp90 cochaperones Cdc37, Sti1, and Sba1 are required for the response to amino acid starvation. Taken together, these data identify Gcn2 as a novel target for Hsp90, which plays a crucial role for the maturation and regulation of Gcn2.

scholarly journals Large G protein α-subunit XLαs limits clathrin-mediated endocytosis and regulates tissue iron levels in vivo

2017 ◽  
Vol 114 (45) ◽  
pp. E9559-E9568 ◽  
Author(s):  
Qing He ◽  
Richard Bouley ◽  
Zun Liu ◽  
Marc N. Wein ◽  
Yan Zhu ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
G Protein ◽  
Critical Role ◽  
Hek293 Cells ◽  
Activity Levels ◽  
Α Subunit ◽  
Proteomic Screen ◽  
Protein Levels ◽  
G Protein Α Subunit

Alterations in the activity/levels of the extralarge G protein α-subunit (XLαs) are implicated in various human disorders, such as perinatal growth retardation. Encoded by GNAS, XLαs is partly identical to the α-subunit of the stimulatory G protein (Gsα), but the cellular actions of XLαs remain poorly defined. Following an initial proteomic screen, we identified sorting nexin-9 (SNX9) and dynamins, key components of clathrin-mediated endocytosis, as binding partners of XLαs. Overexpression of XLαs in HEK293 cells inhibited internalization of transferrin, a process that depends on clathrin-mediated endocytosis, while its ablation by CRISPR/Cas9 in an osteocyte-like cell line (Ocy454) enhanced it. Similarly, primary cardiomyocytes derived from XLαs knockout (XLKO) pups showed enhanced transferrin internalization. Early postnatal XLKO mice showed a significantly higher degree of cardiac iron uptake than wild-type littermates following iron dextran injection. In XLKO neonates, iron and ferritin levels were elevated in heart and skeletal muscle, where XLαs is normally expressed abundantly. XLKO heart and skeletal muscle, as well as XLKO Ocy454 cells, showed elevated SNX9 protein levels, and siRNA-mediated knockdown of SNX9 in XLKO Ocy454 cells prevented enhanced transferrin internalization. In transfected cells, XLαs also inhibited internalization of the parathyroid hormone and type 2 vasopressin receptors. Internalization of transferrin and these G protein-coupled receptors was also inhibited in cells expressing an XLαs mutant missing the Gα portion, but not Gsα or an N-terminally truncated XLαs mutant unable to interact with SNX9 or dynamin. Thus, XLαs restricts clathrin-mediated endocytosis and plays a critical role in iron/transferrin uptake in vivo.

scholarly journals Buprenorphine Metabolites, Buprenorphine-3-glucuronide and Norbuprenorphine-3-glucuronide, Are Biologically Active

2011 ◽  
Vol 115 (6) ◽  
pp. 1251-1260 ◽  
Author(s):  
Sarah M. Brown ◽  
Michael Holtzman ◽  
Thomas Kim ◽  
Evan D. Kharasch
Keyword(s):  
Radioligand Binding ◽  
Competitive Inhibition ◽  
Antinociceptive Effect ◽  
Plasma Concentrations ◽  
Parent Drug ◽  
Biologically Active ◽  
Whole Body ◽  
Tail Flick ◽  
High Affinity

Background The long-lasting high-affinity opioid buprenorphine has complex pharmacology, including ceiling effects with respect to analgesia and respiratory depression. Plasma concentrations of the major buprenorphine metabolites norbuprenorphine, buprenorphine-3-glucuronide, and norbuprenorphine-3-glucuronide approximate or exceed those of the parent drug. Buprenorphine glucuronide metabolites pharmacology is undefined. This investigation determined binding and pharmacologic activity of the two glucuronide metabolites, and in comparison with buprenorphine and norbuprenorphine. Methods Competitive inhibition of radioligand binding to human μ, κ, and δ opioid and nociceptin receptors was used to determine glucuronide binding affinities for these receptors. Common opiate effects were assessed in vivo in SwissWebster mice. Antinociception was assessed using a tail-flick assay, respiratory effects were measured using unrestrained whole-body plethysmography, and sedation was assessed by inhibition of locomotion measured by open-field testing. Results Buprenorphine-3-glucuronide had high affinity for human μ (Ki [inhibition constant] = 4.9 ± 2.7 pM), δ (Ki = 270 ± 0.4 nM), and nociceptin (Ki = 36 ± 0.3 μM) but not κ receptors. Norbuprenorphine-3-glucuronide had affinity for human κ (Ki = 300 ± 0.5 nM) and nociceptin (Ki = 18 ± 0.2 μM) but not μ or δ receptors. At the dose tested, buprenorphine-3-glucuronide had a small antinociceptive effect. Neither glucuronide had significant effects on respiratory rate, but norbuprenorphine-3-glucuronide decreased tidal volume. Norbuprenorphine-3-glucuronide also caused sedation. Conclusions Both glucuronide metabolites of buprenorphine are biologically active at doses relevant to metabolite exposures, which occur after buprenorphine. Activity of the glucuronides may contribute to the overall pharmacology of buprenorphine.

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