%0 Journal Article
%A Schneider, K.
%A Dimroth, B. M.
%A Bott, M.
%T Biosynthesis of the prosthetic group of citrate lyase
%J Biochemistry
%V 39
%@ 0006-2960
%C Columbus, Ohio
%I American Chemical Society
%M PreJuSER-26938
%P 9438 - 9450
%D 2000
%Z Record converted from VDB: 12.11.2012
%X Citrate 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.
%K Acyl Carrier Protein: biosynthesis
%K Amino Acid Sequence
%K Apoproteins: biosynthesis
%K Carbon-Sulfur Ligases: biosynthesis
%K Carbon-Sulfur Ligases: genetics
%K Coenzyme A: biosynthesis
%K Enzyme Precursors: metabolism
%K Escherichia coli: enzymology
%K Escherichia coli: genetics
%K Genes, Bacterial
%K Molecular Sequence Data
%K Multienzyme Complexes: biosynthesis
%K Multienzyme Complexes: chemistry
%K Multienzyme Complexes: genetics
%K Multigene Family
%K Operon
%K Oxo-Acid-Lyases: biosynthesis
%K Oxo-Acid-Lyases: chemistry
%K Oxo-Acid-Lyases: genetics
%K Structure-Activity Relationship
%K Acyl Carrier Protein (NLM Chemicals)
%K Apoproteins (NLM Chemicals)
%K Enzyme Precursors (NLM Chemicals)
%K Multienzyme Complexes (NLM Chemicals)
%K dephosphocoenzyme A (NLM Chemicals)
%K Coenzyme A (NLM Chemicals)
%K Oxo-Acid-Lyases (NLM Chemicals)
%K citrate (pro-3S)-lyase (NLM Chemicals)
%K Carbon-Sulfur Ligases (NLM Chemicals)
%K long-chain-fatty-acid-(acyl-carrier-protein) ligase (NLM Chemicals)
%K citrate (pro-3S)-lyase ligase (NLM Chemicals)
%K J (WoSType)
%F PUB:(DE-HGF)16
%9 Journal Article
%$ pmid:10924139
%U <Go to ISI:>//WOS:000088593300042
%U https://juser.fz-juelich.de/record/26938