Detection of bacterial lipoic acid. A modified gas-chromatographic-mass-spectrometric procedure

Pratt, Keynan; Carles, Christophe; Carne, T. J.; Danson, Michael J.; Stevenson, Kenneth J.

doi:10.1042/bj2580749

Cited by 33 publications

(14 citation statements)

References 21 publications

(25 reference statements)

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“…As for the separation assay, cellular LA was determined by GC-FPD (flame photometric detection) and GC-MS (mass spectrometry) after hydrolytic cleavage of the protein-bound LA by concentrated acid or base at high temperature [25][26][27][28]. Since the GC methods included the strong acid and base hydrolysis of the proteins containing LA, the resulting amounts were the sum of the protein-bound LA and free LA (non-protein LA) in the sample.…”

Section: Introductionmentioning

confidence: 99%

Selective and sensitive determination of lipoyllysine (protein-bound α-lipoic acid) in biological specimens by high-performance liquid chromatography with fluorescence detection

Satoh

Shindoh

Min

et al. 2008

Analytica Chimica Acta

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Section: Introductionmentioning

confidence: 99%

Selective and sensitive determination of lipoyllysine (protein-bound α-lipoic acid) in biological specimens by high-performance liquid chromatography with fluorescence detection

Satoh

Shindoh

Min

et al. 2008

Analytica Chimica Acta

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“…Temperature was later increased to 270°C at a rate of 10 degrees/ min. The gas chromatograms were searched for the presence of methyl octanoate from the library or extracted for methyl lipoate ions obtained from standards (19).…”

Section: Acid Hydrolysis and Gc-ms Of Lipb Extracts-preparationsmentioning

confidence: 99%

Protein-Protein Interactions in Assembly of Lipoic Acid on the 2-Oxoacid Dehydrogenases of Aerobic Metabolism

Hassan¹,

Cronan

2011

Journal of Biological Chemistry

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Lipoic acid is a covalently attached cofactor essential for the activity of 2-oxoacid dehydrogenases and the glycine cleavage system. In the absence of lipoic acid modification, the dehydrogenases are inactive, and aerobic metabolism is blocked. In Escherichia coli, two pathways for the attachment of lipoic acid exist, a de novo biosynthetic pathway dependent on the activities of the LipB and LipA proteins and a lipoic acid scavenging pathway catalyzed by the LplA protein. LipB is responsible for octanoylation of the E2 components of 2-oxoacid dehydrogenases to provide the substrates of LipA, an S-adenosyl-L-methionine radical enzyme that inserts two sulfur atoms into the octanoyl moiety to give the active lipoylated dehydrogenase complexes. We report that the intact pyruvate and 2-oxoglutarate dehydrogenase complexes specifically copurify with both LipB and LipA. Proteomic, genetic, and dehydrogenase activity data indicate that all of the 2-oxoacid dehydrogenase components are present. In contrast, LplA, the lipoate protein ligase enzyme of lipoate salvage, shows no interaction with the 2-oxoacid dehydrogenases. The interaction is specific to the dehydrogenases in that the third lipoic acid-requiring enzyme of Escherichia coli, the glycine cleavage system H protein, does not copurify with either LipA or LipB. Studies of LipB interaction with engineered variants of the E2 subunit of 2-oxoglutarate dehydrogenase indicate that binding sites for LipB reside both in the lipoyl domain and catalytic core sequences. We also report that LipB forms a very tight, albeit noncovalent, complex with acyl carrier protein. These results indicate that lipoic acid is not only assembled on the dehydrogenase lipoyl domains but that the enzymes that catalyze the assembly are also present "on site." Lipoic acid ((R)-5-(1,2-dithiolan-3-yl) pentanoic acid) is a sulfur-containing cofactor covalently attached to and essential for the function of several key enzymatic complexes of central metabolism. These include the pyruvate dehydrogenase (PDH), 2 2-oxoglutarate dehydrogenase (OGDH), branched-chain 2-oxoacid dehydrogenase, acetoin dehydrogenase, and the glycine cleavage system (GCV) (1). Escherichia coli contains only the PDH, OGDH, and GCV enzyme complexes, the first two of which are essential for aerobic metabolism. In each of these enzymes, a specific component (E2 in the 2-oxoacid dehydrogenases, H protein in the glycine cleavage system) is modified by the covalent attachment of lipoic acid to the ⑀-amino group of a conserved lysine residue via an amide bond. The lysine residues protrude from the surfaces of the highly conserved domains, which are generally called lipoyl domains (LD). The LD-bound coenzyme shuttles reaction intermediates among the multiple active sites of these multiprotein complexes in a type of covalent substrate channeling (1).PDH catalyzes the oxidative decarboxylation of pyruvate to form acetyl-CoA (Fig. 1) (1). This very large enzyme complex (larger than the ribosome) consists of multiple copies of three compo...

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“…No OADHC activity has ever been detected in any archaeon [5]. However, detection of E3 and lipoic acid in various archaea [10–12] led to the discovery of a putative OADHC operon in Haloferax volcanii [13] and our subsequent detection of similar putative operons in the genome sequences of the aerobic archaea Thermoplasma acidophilum, S. solfataricus, Sulfolobus acidocaldarius , A. pernix, Pyrobaculum aerophilum, Halobacterium NRC1 and Ferroplasma acidophilum (M. G. Posner, unpublished data).…”

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The 2‐oxoacid dehydrogenase multi‐enzyme complex of the archaeon Thermoplasma acidophilum − recombinant expression, assembly and characterization

Heath¹,

Posner²,

Aass³

et al. 2007

The FEBS Journal

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In aerobic bacteria and eukaryotes, a family of 2-oxoacid dehydrogenase multi-enzyme complexes (OADHCs) functions in the pathways of central metabolism. The complexes are responsible for the oxi-dative decarboxylation of 2-oxoacids to their corresponding acyl-CoAs. Members of the family include the pyruvate dehydrogenase complex (PDHC), which catalyzes the conversion of pyruvate to acetyl-CoA and so links glycolysis and the citric acid cycle; the 2-oxoglutarate dehydrogenase complex (OGDHC), which catalyzes the conversion of 2-oxoglutarate to succinyl-CoA within the citric acid cycle; and the branched-chain 2-oxoacid dehydrogenase complex (BCOADHC), which oxidatively decarboxylates the branched-chain 2-oxoacids produced by the transami-nation of valine, leucine and isoleucine. The complexes comprise multiple copies of three component enzymes: 2-oxoacid decarboxylase (E1), dihydrolipoyl acyl-trans-ferase (E2) and dihydrolipoamide dehydrogenase (E3) [1-3]. E2 forms the structural core of the complex, with multiple polypeptide chains associating into octa-hedral (24-mer) or icosahedral (60-mer) configurations, depending on the particular complex and the source organism [2,4]. E1 and E3 bind noncovalently to the Keywords Archaea; metabolism; multi-enzyme complex; 2-oxoacid dehydrogenase; thermophile Correspondence M. J. The aerobic archaea possess four closely spaced, adjacent genes that encode proteins showing significant sequence identities with the bacterial and eukaryal components comprising the 2-oxoacid dehydrogenase multi-enzyme complexes. However, catalytic activities of such complexes have never been detected in the archaea, although 2-oxoacid ferredoxin oxidore-ductases that catalyze the equivalent metabolic reactions are present. In the current paper, we clone and express the four genes from the thermophilic archaeon, Thermoplasma acidophilum, and demonstrate that the recombi-nant enzymes are active and assemble into a large (M r ¼ 5 · 10 6) multi-enzyme complex. The post-translational incorporation of lipoic acid into the transacylase component of the complex is demonstrated, as is the assembly of this enzyme into a 24-mer core to which the other components bind to give the functional multi-enzyme system. This assembled complex is shown to catalyze the oxidative decarboxylation of branched-chain 2-oxoacids and pyruvate to their corresponding acyl-CoA derivatives. Our data constitute the first proof that the archaea possess a functional 2-oxo-acid dehydrogenase complex. Abbreviations BCOADHC, branched-chain 2-oxoacid dehydrogenase complex; CoASH, coenzyme-A; DLS, dynamic light scattering; E1, 2-oxoacid decarboxylase; E2, dihydrolipoyl acyl-transferase; E3, dihydrolipoamide dehydrogenase; FOR, ferredoxin oxidoreductase; IPTG, isopropyl thio-b-D-galactoside; M r , relative molecular mass; OADHC, 2-oxoacid dehydrogenase complex; OGDHC, 2-oxoglutarate dehydrogenase multienzyme complex; PDHC, pyruvate dehydrogenase complex; TPP, thiamine pyrophosphate.

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Detection of bacterial lipoic acid. A modified gas-chromatographic-mass-spectrometric procedure

Cited by 33 publications

References 21 publications

Selective and sensitive determination of lipoyllysine (protein-bound α-lipoic acid) in biological specimens by high-performance liquid chromatography with fluorescence detection

Selective and sensitive determination of lipoyllysine (protein-bound α-lipoic acid) in biological specimens by high-performance liquid chromatography with fluorescence detection

Protein-Protein Interactions in Assembly of Lipoic Acid on the 2-Oxoacid Dehydrogenases of Aerobic Metabolism

The 2‐oxoacid dehydrogenase multi‐enzyme complex of the archaeon Thermoplasma acidophilum − recombinant expression, assembly and characterization

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