AccD1 And AccA1 from M. tuberculosis form A dodecameric MCC-type holo complex

Mycobacteria have an unusual redundancy of six putative carboxyltransferase genes that form high-molecular weight holo acyl coenzyme A carboxylase complexes with a complementary set of three biotin carboxylase genes. Most of these enzyme complexes use small fatty acid coenzyme A esters as substrate, to allow their extension by one methylene group via a carboxybiotin-mediated α-carboxylation reaction. Redundant occurrence of these complexes was assumed to be related to highly complex enzymatic requirements in lipid biosynthesis, as the mycobacterial thick cell wall comprises unusual very long chain fatty acids, including mycolic acid. We have solved two high-resolution crystal structures of the 350 kDa hexameric assemblies of two different acyl coenzyme A carboxylase hexameric assemblies, AccD5 and AccD6 [1; Anandhakrishnan et al., unpublished], and characterized these enzyme complexes functionally. In a second step we investigated the acyl coenzyme A carboxylase complex AccD1-AccA1 from Mycobacteria tuberculosis with hitherto unknown function. By using a metabolomics approach we found that AccD1-AccA1 is involved in branched amino acid catabolism, which was not investigated in mycobacteria before [Ehebauer et al, unpublished. Using an in vitro assay, we show that the enzyme complex uses methylcrotonyl coenzyme A as substrate]. We determined the overall architecture of the 700 kDa AccD1-AccA1 complex to be formed from three layers of a central AccD1 hexameric ring, flanked by two distal tiers composed of three AccA1 subunits each. Our electron microscopy data match the overall dimensions of a methylcrotonyl coenzyme A holo complex with known structure and thus support our functional findings. Our data suggest a unique functional role of the AccD1-AccA1 complex within the Mycobacterium tuberculosis acyl coenzyme A carboxylase interactome. Ultimately, it is our goal to solve this and related structures of ACCase holo complexes by high-resolution crystallography as well. The abstract is dedicated to Louis Delbaere with whom I shared time during my PhD at the University of Basel, Switzerland.

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Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus

Molecular and Cellular Biology ◽

10.1128/mcb.9.1.83-91.1989 ◽

1989 ◽

Vol 9 (1) ◽

pp. 83-91

Author(s):

S Miyazawa ◽

T Osumi ◽

T Hashimoto ◽

K Ohno ◽

S Miura ◽

...

Keyword(s):

Amino Acid ◽

Rat Liver ◽

Coenzyme A ◽

Terminal Region ◽

Proteinase K ◽

Amino Acid Residues ◽

C Terminus ◽

Targeting Signal ◽

Acyl Coenzyme A

To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experiments with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX residues at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.

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Pseudomonas aeruginosa PqsA Is an Anthranilate-Coenzyme A Ligase

Journal of Bacteriology ◽

10.1128/jb.01140-07 ◽

2007 ◽

Vol 190 (4) ◽

pp. 1247-1255 ◽

Cited By ~ 86

Author(s):

James P. Coleman ◽

L. Lynn Hudson ◽

Susan L. McKnight ◽

John M. Farrow ◽

M. Worth Calfee ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Coenzyme A ◽

Intercellular Signaling ◽

Signaling Systems ◽

Coa Ligase ◽

Coenzyme A Ligase ◽

Opportunistic Human Pathogen ◽

Acyl Coenzyme A

ABSTRACT Pseudomonas aeruginosa is an opportunistic human pathogen which relies on several intercellular signaling systems for optimum population density-dependent regulation of virulence genes. The Pseudomonas quinolone signal (PQS) is a 3-hydroxy-4-quinolone with a 2-alkyl substitution which is synthesized by the condensation of anthranilic acid with a 3-keto-fatty acid. The pqsABCDE operon has been identified as being necessary for PQS production, and the pqsA gene encodes a predicted protein with homology to acyl coenzyme A (acyl-CoA) ligases. In order to elucidate the first step of the 4-quinolone synthesis pathway in P. aeruginosa, we have characterized the function of the pqsA gene product. Extracts prepared from Escherichia coli expressing PqsA were shown to catalyze the formation of anthraniloyl-CoA from anthranilate, ATP, and CoA. The PqsA protein was purified as a recombinant His-tagged polypeptide, and this protein was shown to have anthranilate-CoA ligase activity. The enzyme was active on a variety of aromatic substrates, including benzoate and chloro and fluoro derivatives of anthranilate. Inhibition of PQS formation in vivo was observed for the chloro- and fluoroanthranilate derivatives, as well as for several analogs which were not PqsA enzymatic substrates. These results indicate that the PqsA protein is responsible for priming anthranilate for entry into the PQS biosynthetic pathway and that this enzyme may serve as a useful in vitro indicator for potential agents to disrupt quinolone signaling in P. aeruginosa.

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Regulation of Rabbit Intestinal Acyl Coenzyme A-Cholesterol Acyltransferase In Vivo and In Vitro

Gastroenterology ◽

10.1016/s0016-5085(82)80019-x ◽

1982 ◽

Vol 83 (4) ◽

pp. 873-880 ◽

Cited By ~ 50

Author(s):

F. Jeffrey Field ◽

Allen D. Cooper ◽

Sandra K. Erickson

Keyword(s):

Coenzyme A ◽

Cholesterol Acyltransferase ◽

Acyl Coenzyme A

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Amelioration of Cryptosporidium parvum Infection In Vitro and In Vivo by Targeting Parasite Fatty Acyl-Coenzyme A Synthetases

The Journal of Infectious Diseases ◽

10.1093/infdis/jit645 ◽

2013 ◽

Vol 209 (8) ◽

pp. 1279-1287 ◽

Cited By ~ 23

Author(s):

F. Guo ◽

H. Zhang ◽

J. M. Fritzler ◽

S. D. Rider ◽

L. Xiang ◽

...

Keyword(s):

Cryptosporidium Parvum ◽

Coenzyme A ◽

Fatty Acyl ◽

Acyl Coenzyme A

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Suspect screening and targeted analysis of acyl coenzyme A thioesters in bacterial cultures using a high-resolution tribrid mass spectrometer

Analytical and Bioanalytical Chemistry ◽

10.1007/s00216-021-03318-3 ◽

2021 ◽

Author(s):

Nevenka Cakić ◽

Bernd Kopke ◽

Ralf Rabus ◽

Heinz Wilkes

Keyword(s):

High Resolution ◽

Mass Spectrometer ◽

Coenzyme A ◽

Neutral Loss ◽

Degradation Pathway ◽

Suspect Screening ◽

Bacterial Cultures ◽

Targeted Analysis ◽

Wide Range ◽

Acyl Coenzyme A

AbstractAnalysis of acyl coenzyme A thioesters (acyl-CoAs) is crucial in the investigation of a wide range of biochemical reactions and paves the way to fully understand the concerned metabolic pathways and their superimposed networks. We developed two methods for suspect screening of acyl-CoAs in bacterial cultures using a high-resolution Orbitrap Fusion tribrid mass spectrometer. The methods rely on specific fragmentation patterns of the target compounds, which originate from the coenzyme A moiety. They make use of the formation of the adenosine 3′,5′-diphosphate key fragment (m/z 428.0365) and the neutral loss of the adenosine 3′-phosphate-5′-diphosphate moiety (506.9952) as preselection criteria for the detection of acyl-CoAs. These characteristic ions are generated either by an optimised in-source fragmentation in a full scan Orbitrap measurement or by optimised HCD fragmentation. Additionally, five different filters are included in the design of method. Finally, data-dependent MS/MS experiments on specifically preselected precursor ions are performed. The utility of the methods is demonstrated by analysing cultures of the denitrifying betaproteobacterium “Aromatoleum” sp. strain HxN1 anaerobically grown with hexanoate. We detected 35 acyl-CoAs in total and identified 24 of them by comparison with reference standards, including all 9 acyl-CoA intermediates expected to occur in the degradation pathway of hexanoate. The identification of additional acyl-CoAs provides insight into further metabolic processes occurring in this bacterium. The sensitivity of the method described allows detecting acyl-CoAs present in biological samples in highly variable abundances.

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Biochemical and Mutational Analyses of AcuA, the Acetyltransferase Enzyme That Controls the Activity of the Acetyl Coenzyme A Synthetase (AcsA) in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.00340-08 ◽

2008 ◽

Vol 190 (14) ◽

pp. 5132-5136 ◽

Cited By ~ 32

Author(s):

Jeffrey G. Gardner ◽

Jorge C. Escalante-Semerena

Keyword(s):

Bacillus Subtilis ◽

Coenzyme A ◽

Effective Means ◽

Wild Type ◽

Modification System ◽

Vitro Assay ◽

Acetyl Coenzyme A ◽

Acetyl Coenzyme

ABSTRACT The acuABC genes of Bacillus subtilis comprise a putative posttranslational modification system. The AcuA protein is a member of the Gcn5-related N-acetyltransferase (GNAT) superfamily, the AcuC protein is a class I histone deacetylase, and the role of the AcuB protein is not known. AcuA controls the activity of acetyl coenzyme A synthetase (AcsA; EC 6.2.1.1) in this bacterium by acetylating residue Lys549. Here we report the kinetic analysis of wild-type and variant AcuA proteins. We contrived a genetic scheme for the identification of AcuA residues critical for activity. Changes at residues H177 and G187 completely inactivated AcuA and led to its rapid turnover. Changes at residues R42 and T169 were less severe. In vitro assay conditions were optimized, and an effective means of inactivating the enzyme was found. The basic kinetic parameters of wild-type and variant AcuA proteins were obtained and compared to those of eukaryotic GNATs. Insights into how the isolated mutations may exert their deleterious effect were investigated by using the crystal structure of an AcuA homolog.

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Araliachinoside A: A New Triterpene Glycoside From Aralia chinensis Leaves

Natural Product Communications ◽

10.1177/1934578x20952756 ◽

2020 ◽

Vol 15 (9) ◽

pp. 1934578X2095275

Author(s):

Pham Hai Yen ◽

Nguyen Thi Cuc ◽

Phan Thi Thanh Huong ◽

Nguyen Xuan Nhiem ◽

Nguyen Thi Hoai ◽

...

Keyword(s):

Mass Spectrometry ◽

Nuclear Magnetic Resonance ◽

High Resolution ◽

Inhibitory Concentration ◽

In Vitro Assay ◽

Ionization Mass ◽

Vitro Assay ◽

Chemical Structures ◽

Hepg2 Cell Lines

From the leaves of Aralia chinensis, 3 oleanane-type triterpene glycosides have been isolated, including 1 new glycoside, 3β,23 -dihydroxyolean-12-ene-28-oic acid 3 -O-β-d-glucopyranosyl-(1→3)- α-l-arabinopyranosyl-(1→3)-β-d-glucuronopyranoside 28 -O-β-d-glucopyranosyl ester (named as araliachinoside A, 1), and 2 known ones, 3β,23 -dihydroxyolean-12-ene-28-oic acid 3 -O-α-l-arabinopyranosyl-(1→3)- β-d-glucuronopyranoside 28 -O-β-d-glucopyronosyl ester (2) and 3β-hydroxyolean-12-ene-28-oic acid 3 -O-β-d-glucurono pyranoside 28 -O-β-d-glucopyronosyl ester (3). Their chemical structures were elucidated by using a combination of high-resolution electrospray ionization-mass spectrometry, 1-dimensional and 2-dimensional nuclear magnetic resonance spectral data, and by comparison with previous literature. Compounds 1-3 displayed cytotoxic activity toward KB and HepG2 cell lines with half-maximal inhibitory concentration values ranging from 8.1 ± 0.1 to 15.7 ± 0.3 µM in in vitro assay.

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Antimalarial pantothenamide metabolites target acetyl–coenzyme A biosynthesis in Plasmodium falciparum

Science Translational Medicine ◽

10.1126/scitranslmed.aas9917 ◽

2019 ◽

Vol 11 (510) ◽

pp. eaas9917 ◽

Cited By ~ 4

Author(s):

Joost Schalkwijk ◽

Erik L. Allman ◽

Patrick A. M. Jansen ◽

Laura E. de Vries ◽

Julie M. J. Verhoef ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Coenzyme A ◽

Mosquito Vector ◽

Malaria Parasites ◽

Humanized Mouse Model ◽

Acetyl Coenzyme A ◽

Block Transmission ◽

Acetyl Coenzyme ◽

Acyl Coenzyme A

Malaria eradication is critically dependent on new therapeutics that target resistant Plasmodium parasites and block transmission of the disease. Here, we report that pantothenamide bioisosteres were active against blood-stage Plasmodium falciparum parasites and also blocked transmission of sexual stages to the mosquito vector. These compounds were resistant to degradation by serum pantetheinases, showed favorable pharmacokinetic properties, and cleared parasites in a humanized mouse model of P. falciparum infection. Metabolomics revealed that coenzyme A biosynthetic enzymes converted pantothenamides into coenzyme A analogs that interfered with parasite acetyl–coenzyme A anabolism. Resistant parasites generated in vitro showed mutations in acetyl–coenzyme A synthetase and acyl–coenzyme A synthetase 11. Introduction and reversion of these mutations in P. falciparum using CRISPR-Cas9 gene editing confirmed the roles of these enzymes in the sensitivity of the malaria parasites to pantothenamides. These pantothenamide compounds with a new mode of action may have potential as drugs against malaria parasites.

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