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000026938 084__ $$2WoS$$aBiochemistry & Molecular Biology
000026938 1001_ $$0P:(DE-HGF)0$$aSchneider, K.$$b0
000026938 245__ $$aBiosynthesis of the prosthetic group of citrate lyase
000026938 260__ $$aColumbus, Ohio$$bAmerican Chemical Society$$c2000
000026938 300__ $$a9438 - 9450
000026938 3367_ $$0PUB:(DE-HGF)16$$2PUB:(DE-HGF)$$aJournal Article
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000026938 440_0 $$0798$$aBiochemistry$$v39$$x0006-2960
000026938 500__ $$aRecord converted from VDB: 12.11.2012
000026938 520__ $$aCitrate lyase (EC 4.1.3.6) catalyzes the cleavage of citrate to acetate and oxaloacetate and is composed of three subunits (alpha, beta, and gamma). The gamma-subunit serves as an acyl carrier protein (ACP) and contains the prosthetic group 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA, which is attached via a phosphodiester linkage to serine-14 in the enzyme from Klebsiella pneumoniae. In this work, we demonstrate by genetic and biochemical studies with citrate lyase of Escherichia coli and K. pneumoniae that the conversion of apo-ACP into holo-ACP is dependent on the two proteins, CitX (20 kDa) and CitG (33 kDa). In the absence of CitX, only apo-ACP was synthesized in vivo, whereas in the absence of CitG, an adenylylated ACP was produced, with the AMP residue attached to serine-14. The adenylyltransferase activity of CitX could be verified in vitro with purified CitX and apo-ACP plus ATP as substrates. Besides ATP, CTP, GTP, and UTP also served as nucleotidyl donors in vitro, showing that CitX functions as a nucleotidyltransferase. The conversion of apo-ACP into holo-ACP was achieved in vitro by incubation of apo-ACP with CitX, CitG, ATP, and dephospho-CoA. ATP could not be substituted with GTP, CTP, UTP, ADP, or AMP. In the absence of CitG or dephospho-CoA, AMP-ACP was formed. Remarkably, it was not possible to further convert AMP-ACP to holo-ACP by subsequent incubation with CitG and dephospho-CoA. This demonstrates that AMP-ACP is not an intermediate during the conversion of apo- into holo-ACP, but results from a side activity of CitX that becomes effective in the absence of its natural substrate. Our results indicate that holo-ACP formation proceeds as follows. First, a prosthetic group precursor [presumably 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA] is formed from ATP and dephospho-CoA in a reaction catalyzed by CitG. Second, holo-ACP is formed from apo-ACP and the prosthetic group precursor in a reaction catalyzed by CitX.
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000026938 650_2 $$2MeSH$$aAcyl Carrier Protein: biosynthesis
000026938 650_2 $$2MeSH$$aAmino Acid Sequence
000026938 650_2 $$2MeSH$$aApoproteins: biosynthesis
000026938 650_2 $$2MeSH$$aCarbon-Sulfur Ligases: biosynthesis
000026938 650_2 $$2MeSH$$aCarbon-Sulfur Ligases: genetics
000026938 650_2 $$2MeSH$$aCoenzyme A: biosynthesis
000026938 650_2 $$2MeSH$$aEnzyme Precursors: metabolism
000026938 650_2 $$2MeSH$$aEscherichia coli: enzymology
000026938 650_2 $$2MeSH$$aEscherichia coli: genetics
000026938 650_2 $$2MeSH$$aGenes, Bacterial
000026938 650_2 $$2MeSH$$aMolecular Sequence Data
000026938 650_2 $$2MeSH$$aMultienzyme Complexes: biosynthesis
000026938 650_2 $$2MeSH$$aMultienzyme Complexes: chemistry
000026938 650_2 $$2MeSH$$aMultienzyme Complexes: genetics
000026938 650_2 $$2MeSH$$aMultigene Family
000026938 650_2 $$2MeSH$$aOperon
000026938 650_2 $$2MeSH$$aOxo-Acid-Lyases: biosynthesis
000026938 650_2 $$2MeSH$$aOxo-Acid-Lyases: chemistry
000026938 650_2 $$2MeSH$$aOxo-Acid-Lyases: genetics
000026938 650_2 $$2MeSH$$aStructure-Activity Relationship
000026938 650_7 $$00$$2NLM Chemicals$$aAcyl Carrier Protein
000026938 650_7 $$00$$2NLM Chemicals$$aApoproteins
000026938 650_7 $$00$$2NLM Chemicals$$aEnzyme Precursors
000026938 650_7 $$00$$2NLM Chemicals$$aMultienzyme Complexes
000026938 650_7 $$03633-59-8$$2NLM Chemicals$$adephosphocoenzyme A
000026938 650_7 $$085-61-0$$2NLM Chemicals$$aCoenzyme A
000026938 650_7 $$0EC 4.1.3.-$$2NLM Chemicals$$aOxo-Acid-Lyases
000026938 650_7 $$0EC 4.1.3.6$$2NLM Chemicals$$acitrate (pro-3S)-lyase
000026938 650_7 $$0EC 6.2.-$$2NLM Chemicals$$aCarbon-Sulfur Ligases
000026938 650_7 $$0EC 6.2.1.20$$2NLM Chemicals$$along-chain-fatty-acid-(acyl-carrier-protein) ligase
000026938 650_7 $$0EC 6.2.1.22$$2NLM Chemicals$$acitrate (pro-3S)-lyase ligase
000026938 650_7 $$2WoSType$$aJ
000026938 7001_ $$0P:(DE-HGF)0$$aDimroth, B. M.$$b1
000026938 7001_ $$0P:(DE-Juel1)128943$$aBott, M.$$b2$$uFZJ
000026938 773__ $$0PERI:(DE-600)1472258-6$$gVol. 39, p. 9438 - 9450$$p9438 - 9450$$q39<9438 - 9450$$tBiochemistry$$v39$$x0006-2960$$y2000
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