scholarly journals Uncovering the Protocatechuate 2,3-Cleavage Pathway Genes

2009 ◽  
Vol 191 (21) ◽  
pp. 6758-6768 ◽  
Author(s):  
Daisuke Kasai ◽  
Toshihiro Fujinami ◽  
Tomokuni Abe ◽  
Kohei Mase ◽  
Yoshihiro Katayama ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Coenzyme A ◽  
Sole Source ◽  
Pcr Analysis ◽  
Acetyl Coenzyme A ◽  
Acetyl Coenzyme ◽  
Reverse Transcription Pcr ◽  
Semialdehyde Dehydrogenase ◽  
Cell Extracts

ABSTRACT Paenibacillus sp. (formerly Bacillus macerans) strain JJ-1b is able to grow on 4-hydroxybenzoate (4HB) as a sole source of carbon and energy and is known to degrade 4HB via the protocatechuate (PCA) 2,3-cleavage pathway. However, none of the genes involved in this pathway have been identified. In this study, we identified and characterized the JJ-1b genes for the 4HB catabolic pathway via the PCA 2,3-cleavage pathway, which consisted of praR and praABEGFDCHI. Based on the enzyme activities of cell extracts of Escherichia coli carrying praI, praA, praH, praB, praC, and praD, these genes were found to code for 4HB 3-hydroxylase, PCA 2,3-dioxygenase, 5-carboxy-2-hydroxymuconate-6-semialdehyde decarboxylase, 2-hydroxymuconate-6-semialdehyde dehydrogenase, 4-oxalocrotonate (OCA) tautomerase, and OCA decarboxylase, respectively, which are involved in the conversion of 4HB into 2-hydroxypenta-2,4-dienoate (HPD). The praE, praF, and praG gene products exhibited 45 to 61% amino acid sequence identity to the corresponding enzymes responsible for the catabolism of HPD to pyruvate and acetyl coenzyme A. The deduced amino acid sequence of praR showed similarity with those of IclR-type transcriptional regulators. Reverse transcription-PCR analysis revealed that praABEGFDCHI constitute an operon, and these genes were expressed during the growth of JJ-1b on 4HB and PCA. praR-praABEGFDCHI conferred the ability to grow on 4HB to E. coli, suggesting that praEGF were functional for the conversion of HPD to pyruvate and acetyl coenzyme A. A promoter analysis suggested that praR encodes a repressor of the pra operon.

scholarly journals ZmaR, a Novel and Widespread Antibiotic Resistance Determinant That Acetylates Zwittermicin A

1999 ◽  
Vol 181 (17) ◽  
pp. 5455-5460 ◽  
Author(s):  
Elizabeth A. Stohl ◽  
Sean F. Brady ◽  
Jon Clardy ◽  
Jo Handelsman
Keyword(s):  
Antibiotic Resistance ◽  
Coenzyme A ◽  
Sequence Similarity ◽  
Covalent Modification ◽  
Resistance Determinant ◽  
Acetyl Coenzyme A ◽  
Acetyl Coenzyme ◽  
Cell Extracts ◽  
Zwittermicin A

ABSTRACT ZmaR is a resistance determinant of unusual abundance in the environment and confers on gram-positive and gram-negative bacteria resistance to zwittermicin A, a novel broad-spectrum antibiotic produced by species of Bacillus. The ZmaR protein has no sequence similarity to proteins of known function; thus, the purpose of the present study was to determine the function of ZmaR in vitro. Cell extracts of E. coli containing zmaR inactivated zwittermicin A by covalent modification. Chemical analysis of inactivated zwittermicin A by 1H NMR, 13C NMR, and high- and low-resolution mass spectrometry demonstrated that the inactivated zwittermicin A was acetylated. Purified ZmaR protein inactivated zwittermicin A, and biochemical assays for acetyltransferase activity with [14C]acetyl coenzyme A demonstrated that ZmaR catalyzes the acetylation of zwittermicin A with acetyl coenzyme A as a donor group, suggesting that ZmaR may constitute a new class of acetyltransferases. Our results allow us to assign a biochemical function to a resistance protein that has no sequence similarity to proteins of known function, contributing fundamental knowledge to the fields of antibiotic resistance and protein function.

scholarly journals Operation of the CO Dehydrogenase/Acetyl Coenzyme A Pathway in both Acetate Oxidation and Acetate Formation by the Syntrophically Acetate-Oxidizing Bacterium Thermacetogenium phaeum

2005 ◽  
Vol 187 (10) ◽  
pp. 3471-3476 ◽  
Author(s):  
Satoshi Hattori ◽  
Alexander S. Galushko ◽  
Yoichi Kamagata ◽  
Bernhard Schink
Keyword(s):  
Gel Electrophoresis ◽  
Coenzyme A ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Acetate Oxidation ◽  
Co Dehydrogenase ◽  
Protein Patterns ◽  
Acetyl Coenzyme A ◽  
Acetyl Coenzyme ◽  
Cell Extracts

ABSTRACT Thermacetogenium phaeum is a homoacetogenic bacterium that can grow on various substrates, such as pyruvate, methanol, or H2/CO2. It can also grow on acetate if cocultured with the hydrogen-consuming methanogenic partner Methanothermobacter thermautotrophicus. Enzyme activities of the CO dehydrogenase/acetyl coenzyme A (CoA) pathway (CO dehydrogenase, formate dehydrogenase, formyl tetrahydrofolate synthase, methylene tetrahydrofolate dehydrogenase) were detected in cell extracts of pure cultures and of syntrophic cocultures. Mixed cell suspensions of T. phaeum and M. thermautotrophicus oxidized acetate rapidly and produced acetate after addition of H2/CO2 after a short time lag. CO dehydrogenase activity staining after native polyacrylamide gel electrophoresis exhibited three oxygen-labile bands which were identical in pure culture and coculture. Protein profiles of T. phaeum cells after sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the strain exhibited basically the same protein patterns in both pure and syntrophic culture. These results indicate that T. phaeum operates the CO dehydrogenase/acetyl-CoA pathway reversibly both in acetate oxidation and in reductive acetogenesis by using the same biochemical apparatus, although it has to couple this pathway to ATP synthesis in different ways.

Effect of Sethoxydim and Haloxyfop on Acetyl-Coenzyme a Carboxylase Activity inFestucaSpecies

Weed Science ◽  
1989 ◽  
Vol 37 (4) ◽  
pp. 512-516 ◽  
Author(s):  
David E. Stoltenberg ◽  
John W. Gronwald ◽  
Donald L. Wyse ◽  
James D. Burton ◽  
David A. Somers ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Tall Fescue ◽  
Coenzyme A ◽  
Carboxylase Activity ◽  
Acetyl Coenzyme A ◽  
Red Fescue ◽  
Acetyl Coenzyme ◽  
Cell Extracts ◽  
Site Of Action ◽  
Acetyl Coenzyme A Carboxylase

In greenhouse studies, the calculated I50(herbicide application resulting in 50% inhibition of shoot regrowth) in tall fescue was approximately 0.004 kg/ha for both sethoxydim and haloxyfop. In red fescue, the I50for sethoxydim and haloxyfop was 9.4 kg/ha and 0.04 kg/ha, respectively. As measured in crude cell extracts of tall fescue, incorporation of14C-acetyl-coenzyme A into fatty acids was inhibited 62 and 71% by 10 μM sethoxydim and 10 μM haloxyfop, respectively. In red fescue, 10 μM haloxyfop inhibited14C-acetyl-CoA incorporation into fatty acids by 29%, whereas 10 μM sethoxydim had no effect. The I50for inhibition of acetyl-coenzyme A carboxylase activity in tall fescue with sethoxydim and haloxyfop was 6.9 and 5.8 μM, respectively. In red fescue the I50for haloxyfop was 118 μM. Sethoxydim concentrations as high as 1 mM had little effect on acetyl-coenzyme A carboxylase activity in red fescue. These results suggest that acetyl-coenzyme A carboxylase is a sensitive site of action for sethoxydim and haloxyfop in tall fescue, and that tolerance to these herbicides in red fescue is due to the presence of a more tolerant form of the enzyme.

scholarly journals Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A inSyntrophus aciditrophicus, a New Twist on ATP Formation

mBio ◽  
2016 ◽  
Vol 7 (4) ◽  
Author(s):  
Kimberly L. James ◽  
Luis A. Ríos-Hernández ◽  
Neil Q. Wofford ◽  
Housna Mouttaki ◽  
Jessica R. Sieber ◽  
...  
Keyword(s):  
Coenzyme A ◽  
Acetate Kinase ◽  
Acetyl Coa ◽  
Acetyl Coenzyme A ◽  
Atp Formation ◽  
Acetyl Coenzyme ◽  
Cell Extracts ◽  
Fermentative Bacteria ◽  
Phosphate Acetyltransferase ◽  
Syntrophus Aciditrophicus

ABSTRACTSyntrophus aciditrophicusis a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation byS. aciditrophicus. However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome ofS. aciditrophicusleaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show thatS. aciditrophicususes AMP-forming, acetyl-CoA synthetase (Acs1) for ATP synthesis from acetyl-CoA.acs1mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, ofS. aciditrophicusgrown in pure culture and coculture. Cell extracts ofS. aciditrophicushad low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified fromS. aciditrophicusand recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) inS. aciditrophicuscells support the operation of Acs1 in the acetate-forming direction. Thus,S. aciditrophicushas a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase.IMPORTANCEBacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA.Syntrophus aciditrophicusapparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as its genome does not have homologs to the genes for phosphate acetyltransferase and acetate kinase. Here, we show thatS. aciditrophicususes an alternative approach, an AMP-forming, acetyl-CoA synthetase, to make ATP from acetyl-CoA. AMP-forming, acetyl-CoA synthetases were previously thought to function only in the activation of acetate to acetyl-CoA.

scholarly journals Cloning and expression of a novel Mu class murine glutathione transferase isoenzyme

2002 ◽  
Vol 366 (3) ◽  
pp. 817-824 ◽  
Author(s):  
Jianxia GUO ◽  
Ludwika ZIMNIAK ◽  
Piotr ZIMNIAK ◽  
John L. ORCHARD ◽  
Shivendra V. SINGH
Keyword(s):  
Small Intestine ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Glutathione Transferase ◽  
Cloning And Expression ◽  
Pcr Analysis ◽  
Amino Acid Residues ◽  
Reverse Transcription Pcr ◽  
Mouse Small Intestine

The present study describes the cDNA cloning, expression and characterization of a novel Mu class murine glutathione transferase (GST) isoenzyme. Screening of a cDNA library from the small intestine of a female A/J mouse using consensus probes derived from Mu class murine GST genes (mGSTM1—mGSTM5) resulted in the isolation of a full-length cDNA clone of a previously unknown Mu class GST gene (designated as mGSTM7). The choice of tissue was based on our previous identification in female A/J mouse small intestine of a potentially novel Mu class GST isoenzyme. The deduced amino acid sequence of mGSTM7, which comprises of 218 amino acid residues, exhibited about 67—78% identity with other Mu class murine GSTs. Recombinant mGSTM7-7 cross-reacted with anti-(GST Mu) antibodies, but not with anti-(GST Alpha) or anti-(GST Pi) antibodies. The pI and the reverse-phase-HPLC elution profile of recombinant mGSTM7-7 were different from those of other Mu class murine GSTs. The substrate specificity of mGSTM7-7 was also different compared with other Mu class murine GSTs. Interestingly, mGSTM7 had a higher identity with the human Mu class isoenzyme hGSTM4 (87% identity and 94% similarity in the amino acid sequence) than with any of the known mouse Mu class GSTs. Specific activities of recombinant mGSTM7-7 and human GSTM4-4 were comparable towards several substrates. For example, similar to hGSTM4-4, recombinant mGSTM7-7 was poorly active in catalysing the GSH conjugation of 1-chloro-2,4-dinitrobenzene and ethacrynic acid, and lacked activity towards 1,2-dichloro-4-nitrobenzene and 1,2-epoxy-3-(p-nitrophenoxy)propane. These results suggested that hGSTM4-4 might be the human counterpart of mouse GSTM7-7. Reverse transcription-PCR analysis using mGSTM7-specific primers revealed that mGSTM7 is widely expressed in tissues of female A/J mice, including liver, forestomach, lung, kidney, colon and spleen.

scholarly journals Molecular Cloning and Characterization of Two Genes for the Biotin Carboxylase and Carboxyltransferase Subunits of Acetyl Coenzyme A Carboxylase in Myxococcus xanthus

2000 ◽  
Vol 182 (19) ◽  
pp. 5462-5469 ◽  
Author(s):  
Yoshio Kimura ◽  
Rina Miyake ◽  
Yushi Tokumasu ◽  
Masayuki Sato
Keyword(s):  
Amino Acid ◽  
Coenzyme A ◽  
Myxococcus Xanthus ◽  
Biotin Carboxylase ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Acetyl Coenzyme A ◽  
Acetyl Coenzyme ◽  
Acid Protein ◽  
Amino Acid Protein

ABSTRACT We have cloned a DNA fragment from a genomic library ofMyxococcus xanthus using an oligonucleotide probe representing conserved regions of biotin carboxylase subunits of acetyl coenzyme A (acetyl-CoA) carboxylases. The fragment contained two open reading frames (ORF1 and ORF2), designated the accB andaccA genes, capable of encoding a 538-amino-acid protein of 58.1 kDa and a 573-amino-acid protein of 61.5 kDa, respectively. The protein (AccA) encoded by the accA gene was strikingly similar to biotin carboxylase subunits of acetyl-CoA and propionyl-CoA carboxylases and of pyruvate carboxylase. The putative motifs for ATP binding, CO2 fixation, and biotin binding were found in AccA. The accB gene was located upstream of theaccA gene, and they formed a two-gene operon. The protein (AccB) encoded by the accB gene showed high degrees of sequence similarity with carboxyltransferase subunits of acetyl-CoA and propionyl-CoA carboxylases and of methylmalonyl-CoA decarboxylase. Carboxybiotin-binding and acyl-CoA-binding domains, which are conserved in several carboxyltransferase subunits of acyl-CoA carboxylases, were found in AccB. An accA disruption mutant showed a reduced growth rate and reduced acetyl-CoA carboxylase activity compared with the wild-type strain. Western blot analysis indicated that the product of the accA gene was a biotinylated protein that was expressed during the exponential growth phase. Based on these results, we propose that this M. xanthus acetyl-CoA carboxylase consists of two subunits, which are encoded by the accB andaccA genes, and occupies a position between prokaryotic and eukaryotic acetyl-CoA carboxylases in terms of evolution.

scholarly journals Coupled Ferredoxin and Crotonyl Coenzyme A (CoA) Reduction with NADH Catalyzed by the Butyryl-CoA Dehydrogenase/Etf Complex from Clostridium kluyveri

2007 ◽  
Vol 190 (3) ◽  
pp. 843-850 ◽  
Author(s):  
Fuli Li ◽  
Julia Hinderberger ◽  
Henning Seedorf ◽  
Jin Zhang ◽  
Wolfgang Buckel ◽  
...  
Keyword(s):  
Energy Metabolism ◽  
Coenzyme A ◽  
Acetyl Coa ◽  
Acetyl Coenzyme A ◽  
Acetyl Coenzyme ◽  
Cell Extracts ◽  
Clostridium Kluyveri ◽  
Fully Coupled ◽  
Coupled Reaction ◽  
Dehydrogenase Complex

ABSTRACT Cell extracts of butyrate-forming clostridia have been shown to catalyze acetyl-coenzyme A (acetyl-CoA)- and ferredoxin-dependent formation of H2 from NADH. It has been proposed that these bacteria contain an NADH:ferredoxin oxidoreductase which is allosterically regulated by acetyl-CoA. We report here that ferredoxin reduction with NADH in cell extracts from Clostridium kluyveri is catalyzed by the butyryl-CoA dehydrogenase/Etf complex and that the acetyl-CoA dependence previously observed is due to the fact that the cell extracts catalyze the reduction of acetyl-CoA with NADH via crotonyl-CoA to butyryl-CoA. The cytoplasmic butyryl-CoA dehydrogenase complex was purified and is shown to couple the endergonic reduction of ferredoxin (E0′ = −410 mV) with NADH (E0′ = −320 mV) to the exergonic reduction of crotonyl-CoA to butyryl-CoA (E0′ = −10 mV) with NADH. The stoichiometry of the fully coupled reaction is extrapolated to be as follows: 2 NADH + 1 oxidized ferredoxin + 1 crotonyl-CoA = 2 NAD+ + 1 ferredoxin reduced by two electrons + 1 butyryl-CoA. The implications of this finding for the energy metabolism of butyrate-forming anaerobes are discussed in the accompanying paper.

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