scholarly journals Evidence that ACN1 (acetate non-utilizing 1) prevents carbon leakage from peroxisomes during lipid mobilization in Arabidopsis seedlings

2011 ◽  
Vol 437 (3) ◽  
pp. 505-513 ◽  
Author(s):  
Elizabeth Allen ◽  
Annick Moing ◽  
Jonathan A. D. Wattis ◽  
Tony Larson ◽  
Mickaël Maucourt ◽  
...  
Keyword(s):  
Carbon Sink ◽  
Glyoxylate Cycle ◽  
Eicosenoic Acid ◽  
Primary Metabolism ◽  
Acetyl Coa ◽  
Primary Metabolite ◽  
Lipid Mobilization ◽  
Transcript Levels ◽  
Chain Acyl ◽  
Acetate Accumulation

ACN1 (acetate non-utilizing 1) is a short-chain acyl-CoA synthetase which recycles free acetate to acetyl-CoA in peroxisomes of Arabidopsis. Pulse-chase [2-13C]acetate feeding of the mutant acn1–2 revealed that acetate accumulation and assimilation were no different to that of wild-type, Col-7. However, the lack of acn1–2 led to a decrease of nearly 50% in 13C-labelling of glutamine, a major carbon sink in seedlings, and large decreases in primary metabolite levels. In contrast, acetyl-CoA levels were higher in acn1–2 compared with Col-7. The disappearance of eicosenoic acid was slightly delayed in acn1–2 indicating only a small effect on the rate of lipid breakdown. A comparison of transcript levels in acn1–2 and Col-7 showed that induced genes included a number of metabolic genes and also a large number of signalling-related genes. Genes repressed in the mutant were represented primarily by embryogenesis-related genes. Transcript levels of glyoxylate cycle genes also were lower in acn1–2 than in Col-7. We conclude that deficiency in peroxisomal acetate assimilation comprises only a small proportion of total acetate use, but this affects both primary metabolism and gene expression. We discuss the possibility that ACN1 safeguards against the loss of carbon as acetate from peroxisomes during lipid mobilization.

Modelling the peroxisomal carbon leak during lipid mobilization in Arabidopsis

2010 ◽  
Vol 38 (5) ◽  
pp. 1230-1233 ◽  
Author(s):  
Mark A. Hooks ◽  
Elizabeth Allen ◽  
Jonathan A.D. Wattis
Keyword(s):  
Carbon Metabolism ◽  
Glyoxylate Cycle ◽  
Seedling Development ◽  
Wild Type ◽  
Acetyl Coa ◽  
Primary Metabolite ◽  
Lipid Mobilization ◽  
Acetate Assimilation ◽  
Development Levels ◽  
The Glyoxylate Cycle

Mutation of the ACN1 (acetate non-utilizing 1) locus of Arabidopsis results in altered acetate assimilation into gluconeogenic sugars and anapleurotic amino acids and leads to an overall depression in primary metabolite levels by approx. 50% during seedling development. Levels of acetyl-CoA were higher in acn1 compared with wild-type, which is counterintuitive to the activity of ACN1 as a peroxisomal acetyl-CoA synthetase. We hypothesize that ACN1 recycles free acetate to acetyl-CoA within peroxisomes in order that carbon remains fed into the glyoxylate cycle. When ACN1 is not present, carbon in the form of acetate can leak out of peroxisomes and is reactivated to acetyl-CoA within the cytosol. Kinetic models incorporating estimates of carbon input and pathway dynamics from a variety of literature sources have proven useful in explaining how ACN1 may prevent the carbon leak and even contribute to the control of peroxisomal carbon metabolism.

The joint acute effect of tetracycline, erythromycin and sulfamethoxazole on acetoclastic methanogens

2015 ◽  
Vol 71 (8) ◽  
pp. 1128-1135 ◽  
Author(s):  
Sevcan Aydın ◽  
Bahar Ince ◽  
Orhan Ince
Keyword(s):  
Mrna Level ◽  
Acute Effect ◽  
Anaerobic Treatment ◽  
Anaerobic Sludge ◽  
Batch Reactors ◽  
Sequencing Batch Reactors ◽  
Acetyl Coa ◽  
Acetate Accumulation ◽  
Acetate Degradation ◽  
Industry Wastewater

In this study, we aimed to develop an understanding of the triple effects of sulfamethoxazole–erythromycin–tetracycline (ETS) and the dual effects of sulfamethoxazole–tetracycline (ST), erythromycin–sulfamethoxazole (ES) and erythromycin–tetracycline (ET) on the anaerobic treatment of pharmaceutical industry wastewater throughout a year of operation. Concentrations of the antibiotics in the influent were gradually increased until the metabolic collapse of the anaerobic sequencing batch reactors (SBRs), which corresponded to ETS (40 + 3 + 3 mg/L) and ST (25 + 2.5 mg/L), ET (4 + 4 mg/L) and ES (3 + 40 mg/L). Acetate accumulation in the anaerobic SBRs, acetoclastic activity of the anaerobic sludge taken from different antibiotic feeding stages and also expression of acetyl-coA synthetase from the acetoclastic methanogenic pathway on the mRNA level were assessed. The results indicated that, while acetate accumulation and decrease of acetoclastic activity were observed after stage 3 in the ST and ES reactors, and stage 7 in the ETS and ET reactors, the expression of acetyl-coA synthetase was mostly decreased in the last stages in all SBRs, in which antibiotic mixture feeding was terminated. It might be speculated that acetoclastic methanogens have an important role in acetate degradation by expressing acetyl-coA synthetase.

scholarly journals The Apparent Malate Synthase Activity of Rhodobacter sphaeroides Is Due to Two Paralogous Enzymes, (3S)-Malyl-Coenzyme A (CoA)/β-Methylmalyl-CoA Lyase and (3S)- Malyl-CoA Thioesterase

2010 ◽  
Vol 192 (5) ◽  
pp. 1249-1258 ◽  
Author(s):  
Tobias J. Erb ◽  
Lena Frerichs-Revermann ◽  
Georg Fuchs ◽  
Birgit E. Alber
Keyword(s):  
Rhodobacter Sphaeroides ◽  
Coenzyme A ◽  
Glyoxylate Cycle ◽  
Condensation Reaction ◽  
Malate Synthase ◽  
Acetyl Coa ◽  
Acetyl Coenzyme A ◽  
Claisen Condensation ◽  
Enzyme Functions ◽  
The Glyoxylate Cycle

ABSTRACT Assimilation of acetyl coenzyme A (acetyl-CoA) is an essential process in many bacteria that proceeds via the glyoxylate cycle or the ethylmalonyl-CoA pathway. In both assimilation strategies, one of the final products is malate that is formed by the condensation of acetyl-CoA with glyoxylate. In the glyoxylate cycle this reaction is catalyzed by malate synthase, whereas in the ethylmalonyl-CoA pathway the reaction is separated into two proteins: malyl-CoA lyase, a well-known enzyme catalyzing the Claisen condensation of acetyl-CoA with glyoxylate and yielding malyl-CoA, and an unidentified malyl-CoA thioesterase that hydrolyzes malyl-CoA into malate and CoA. In this study the roles of Mcl1 and Mcl2, two malyl-CoA lyase homologs in Rhodobacter sphaeroides, were investigated by gene inactivation and biochemical studies. Mcl1 is a true (3S)-malyl-CoA lyase operating in the ethylmalonyl-CoA pathway. Notably, Mcl1 is a promiscuous enzyme and catalyzes not only the condensation of acetyl-CoA and glyoxylate but also the cleavage of β-methylmalyl-CoA into glyoxylate and propionyl-CoA during acetyl-CoA assimilation. In contrast, Mcl2 was shown to be the sought (3S)-malyl-CoA thioesterase in the ethylmalonyl-CoA pathway, which specifically hydrolyzes (3S)-malyl-CoA but does not use β-methylmalyl-CoA or catalyze a lyase or condensation reaction. The identification of Mcl2 as thioesterase extends the enzyme functions of malyl-CoA lyase homologs that have been known only as “Claisen condensation” enzymes so far. Mcl1 and Mcl2 are both related to malate synthase, an enzyme which catalyzes both a Claisen condensation and thioester hydrolysis reaction.

scholarly journals Peroxysomal Carnitine Acetyl Transferase Influences Host Colonization Capacity in Sclerotinia sclerotiorum

2013 ◽  
Vol 26 (7) ◽  
pp. 768-780 ◽  
Author(s):  
D. Liberti ◽  
J. A. Rollins ◽  
K. F. Dobinson
Keyword(s):  
Fatty Acids ◽  
Oxalic Acid ◽  
Sclerotinia Sclerotiorum ◽  
Magnaporthe Grisea ◽  
Glyoxylate Cycle ◽  
Wild Type ◽  
Transcript Accumulation ◽  
Acetyl Coa ◽  
Host Colonization ◽  
Carnitine Acetyl Transferase

In lower eukaryotes, the glyoxylate cycle allows cells to utilize two-carbon compounds when simple sugars are not available. In filamentous fungi, glyoxylate metabolism is coupled with β-oxidation of fatty acids, and both are localized to ubiquitous eukaryotic organelles called peroxisomes. Acetyl coenzyme A (acetyl-CoA) produced during β-oxidation is transported via the cytosol into mitochondria for further metabolism. A peroxisomal-specific pathway for acetyl-CoA transport requiring peroxisomal carnitine acetyl transferase (CAT) activity has been identified in Magnaporthe grisea peroxisomes. Here, we report that a Sclerotinia sclerotiorum ortholog of the M. grisea peroxisomal CAT-encoding gene Pth2 (herein designated Ss-pth2) is required for virulence-associated host colonization. Null (ss-pth2) mutants, obtained by in vitro transposon mutagenesis, failed to utilize fatty acids, acetate, or glycerol as sole carbon sources for growth. Gene expression analysis of these mutants showed altered levels of transcript accumulation for glyoxylate cycle enzymes. Ss-pth2 disruption also affected sclerotial, apothecial, and appressorial development and morphology, as well as oxalic acid accumulation when cultured with acetate or oleic acid as sole carbon nutrient sources. Although mutants were able to penetrate and initially colonize host tissue, subsequent colonization was impaired. Genetic complementation with the wild-type Ss-pth2 restored wild-type virulence phenotypes. These findings suggest an essential role in S. sclerotiorum for the peroxisomal metabolic pathways for oxalic acid synthesis and host colonization.

scholarly journals Domestication of the novel alcohologenic acetogen Clostridium sp. AWRP: from isolation to characterization for syngas fermentation

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Joungmin Lee ◽  
Jin Woo Lee ◽  
Cheol Gi Chae ◽  
Soo Jae Kwon ◽  
Yun Jae Kim ◽  
...  
Keyword(s):  
Value Added ◽  
The Novel ◽  
Primary Metabolite ◽  
Alcohol Production ◽  
Waste Gas ◽  
Batch Bioreactor ◽  
Genome Wide ◽  
Acetate Accumulation ◽  
Close Relatives ◽  
Oxidoreduction Potential

Abstract Background Gas-fermenting acetogens have received a great deal of attention for their ability to grow on various syngas and waste gas containing carbon monoxide (CO), producing acetate as the primary metabolite. Among them, some Clostridium species, such as C. ljungdahlii and C. autoethanogenum, are of particular interest as they produce fuel alcohols as well. Despite recent efforts, alcohol production by these species is still unsatisfactory due to their low productivity and acetate accumulation, necessitating the isolation of strains with better phenotypes. Results In this study, a novel alcohol-producing acetogen (Clostridium sp. AWRP) was isolated, and its complete genome was sequenced. This bacterium belongs the same phylogenetic group as C. ljungdahlii, C. autoethanogenum, C. ragsdalei, and C. coskatii based on 16S rRNA homology; however, the levels of genome-wide average nucleotide identity (gANI) for strain AWRP compared with these strains range between 95 and 96%, suggesting that this strain can be classified as a novel species. In addition, strain AWRP produced a substantial amount of ethanol (70–90 mM) from syngas in batch serum bottle cultures, which was comparable to or even exceeded the typical values obtained using its close relatives cultivated under similar conditions. In a batch bioreactor, strain AWRP produced 119 and 12 mM of ethanol and 2,3-butanediol, respectively, while yielding only 1.4 mM of residual acetate. Interestingly, the alcohologenesis of this strain was strongly affected by oxidoreduction potential (ORP), which has not been reported with other gas-fermenting clostridia. Conclusion Considering its ethanol production under low oxidoreduction potential (ORP) conditions, Clostridium sp. AWRP will be an interesting host for biochemical studies to understand the physiology of alcohol-producing acetogens, which will contribute to metabolic engineering of those strains for the production of alcohols and other value-added compounds from syngas.

scholarly journals Common metabolic networks contribute to carbon sink strength of sorghum internodes: implications for bioenergy improvement

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Yin Li ◽  
Min Tu ◽  
Yaping Feng ◽  
Wenqin Wang ◽  
Joachim Messing
Keyword(s):  
Candidate Genes ◽  
Sweet Sorghum ◽  
Metabolic Networks ◽  
Carbon Allocation ◽  
Carbon Sink ◽  
Sink Strength ◽  
Primary Metabolism ◽  
Data Sets ◽  
Rna Seq ◽  
The Common

Abstract Background Sorghum bicolor (L.) is an important bioenergy source. The stems of sweet sorghum function as carbon sinks and accumulate large amounts of sugars and lignocellulosic biomass and considerable amounts of starch, therefore providing a model of carbon allocation and accumulation for other bioenergy crops. While omics data sets for sugar accumulation have been reported in different genotypes, the common features of primary metabolism in sweet genotypes remain unclear. To obtain a cohesive and comparative picture of carbohydrate metabolism between sorghum genotypes, we compared the phenotypes and transcriptome dynamics of sugar-accumulating internodes among three different sweet genotypes (Della, Rio, and SIL-05) and two non-sweet genotypes (BTx406 and R9188). Results Field experiments showed that Della and Rio had similar dynamics and internode patterns of sugar concentration, albeit distinct other phenotypes. Interestingly, cellulose synthases for primary cell wall and key genes in starch synthesis and degradation were coordinately upregulated in sweet genotypes. Sweet sorghums maintained active monolignol biosynthesis compared to the non-sweet genotypes. Comparative RNA-seq results support the role of candidate Tonoplast Sugar Transporter gene (TST), but not the Sugars Will Eventually be Exported Transporter genes (SWEETs) in the different sugar accumulations between sweet and non-sweet genotypes. Conclusions Comparisons of the expression dynamics of carbon metabolic genes across the RNA-seq data sets identify several candidate genes with contrasting expression patterns between sweet and non-sweet sorghum lines, including genes required for cellulose and monolignol synthesis (CesA, PTAL, and CCR), starch metabolism (AGPase, SS, SBE, and G6P-translocator SbGPT2), and sucrose metabolism and transport (TPP and TST2). The common transcriptome features of primary metabolism identified here suggest the metabolic networks contributing to carbon sink strength in sorghum internodes, prioritize the candidate genes for manipulating carbon allocation with bioenergy purposes, and provide a comparative and cohesive picture of the complexity of carbon sink strength in sorghum stem.

scholarly journals Expression and characterization of recombinant fungal acetyl-CoA carboxylase and isolation of a soraphen-binding domain

2004 ◽  
Vol 380 (1) ◽  
pp. 105-110 ◽  
Author(s):  
Stephanie C. WEATHERLY ◽  
Sandra L. VOLRATH ◽  
Tedd D. ELICH
Keyword(s):  
Magnaporthe Grisea ◽  
Fatty Acid Biosynthesis ◽  
Recombinant Expression ◽  
Phytopathogenic Fungi ◽  
Full Length ◽  
Primary Metabolism ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
E Coli ◽  
High Affinity

Acetyl-CoA carboxylase (ACC) catalyses the first step in fatty-acid biosynthesis. Owing to its role in primary metabolism, ACC has been exploited as a commercial herbicide target and identified as a chemically validated fungicide target. In animals, ACC is also a key regulator of fat metabolism. This function has made ACC a prime target for the development of anti-obesity and anti-Type II diabetes therapeutics. Despite its economic importance, there is a lack of published information on recombinant expression of ACC. We report here the expression of enzymically active fungal (Ustilago maydis) ACC in Escherichia coli. The recombinant enzyme exhibited Km values of 0.14±0.013 mM and 0.19±0.041 mM for acetyl-CoA and ATP respectively, which are comparable with those reported for the endogenous enzyme. The polyketide natural product soraphen is a potent inhibitor of the BC (biotin carboxylase) domain of endogenous fungal ACC. Similarly, recombinant ACC activity was inhibited by soraphen with a Ki of 2.1±0.9 nM. A truncated BC domain that included amino acids 2–560 of the full-length protein was also expressed in E. coli. The isolated BC domain was expressed to higher levels, and was more stable than full-length ACC. Although incapable of enzymic turnover, the BC domain exhibited high-affinity soraphen binding (Kd 1.1±0.3 nM), demonstrating a native conformation. Additional BC domains from the phytopathogenic fungi Magnaporthe grisea and Phytophthora infestans were also cloned and expressed, and were shown to exhibit high-affinity soraphen binding. Together, these reagents will be useful for structural studies and assay development.

scholarly journals AMP-forming acetyl-CoA synthetases in Archaea show unexpected diversity in substrate utilization

Archaea ◽  
2006 ◽  
Vol 2 (2) ◽  
pp. 95-107 ◽  
Author(s):  
Cheryl Ingram-Smith ◽  
Kerry S. Smith
Keyword(s):  
Phylogenetic Analysis ◽  
Molecular Basis ◽  
Catalytic Efficiency ◽  
Adenosine Monophosphate ◽  
Archaeoglobus Fulgidus ◽  
Acetyl Coa ◽  
Chain Acyl ◽  
Key Enzyme ◽  
Methanothermobacter Thermautotrophicus ◽  
Branched Chain

Adenosine monophosphate (AMP)-forming acetyl-CoA synthetase (ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1) is a key enzyme for conversion of acetate to acetyl-CoA, an essential intermediate at the junction of anabolic and catabolic pathways. Phylogenetic analysis of putative short and medium chain acyl-CoA synthetase sequences indicates that the ACSs form a distinct clade from other acyl-CoA synthetases. Within this clade, the archaeal ACSs are not monophyletic and fall into three groups composed of both bacterial and archaeal sequences. Kinetic analysis of two archaeal enzymes, an ACS fromMethanothermobacter thermautotrophicus(designated as MT-ACS1) and an ACS fromArchaeoglobus fulgidus(designated as AF-ACS2), revealed that these enzymes have very different properties. MT-ACS1 has nearly 11-fold higher affinity and 14-fold higher catalytic efficiency with acetate than with propionate, a property shared by most ACSs. However, AF-ACS2 has only 2.3-fold higher affinity and catalytic efficiency with acetate than with propionate. This enzyme has an affinity for propionate that is almost identical to that of MT-ACS1 for acetate and nearly tenfold higher than the affinity of MT-ACS1 for propionate. Furthermore, MT-ACS1 is limited to acetate and propionate as acyl substrates, whereas AF-ACS2 can also utilize longer straight and branched chain acyl substrates. Phylogenetic analysis, sequence alignment and structural modeling suggest a molecular basis for the altered substrate preference and expanded substrate range of AF-ACS2 versus MT-ACS1.

scholarly journals Kinetic, Structural, and Mutational Analysis of Acyl-CoA Carboxylase From Thermobifida fusca YX

2021 ◽  
Vol 7 ◽  
Author(s):  
Kiran-Kumar Shivaiah ◽  
Bryon Upton ◽  
Basil J. Nikolau
Keyword(s):  
Substrate Specificity ◽  
Quaternary Structure ◽  
Mutational Analysis ◽  
Streptomyces Coelicolor ◽  
Thermobifida Fusca ◽  
Substrate Selectivity ◽  
Acetyl Coa ◽  
Catalytic Subunits ◽  
Chain Acyl

Acyl-CoA carboxylases (AcCCase) are biotin-dependent enzymes that are capable of carboxylating more than one short chain acyl-CoA substrate. We have conducted structural and kinetic analyses of such an AcCCase from Thermobifida fusca YX, which exhibits promiscuity in carboxylating acetyl-CoA, propionyl-CoA, and butyryl-CoA. The enzyme consists of two catalytic subunits (TfAcCCA and TfAcCCB) and a non-catalytic subunit, TfAcCCE, and is organized in quaternary structure with a A6B6E6 stoichiometry. Moreover, this holoenzyme structure appears to be primarily assembled from two A3 and a B6E6 subcomplexes. The role of the TfAcCCE subunit is to facilitate the assembly of the holoenzyme complex, and thereby activate catalysis. Based on prior studies of an AcCCase from Streptomyces coelicolor, we explored whether a conserved Asp residue in the TfAcCCB subunit may have a role in determining the substrate selectivity of these types of enzymes. Mutating this D427 residue resulted in alterations in the substrate specificity of the TfAcCCase, increasing proficiency for carboxylating acetyl-CoA, while decreasing carboxylation proficiency with propionyl-CoA and butyryl-CoA. Collectively these results suggest that residue D427 of AcCCB subunits is an important, but not sole determinant of the substrate specificity of AcCCase enzymes.

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