Biosynthesis of the prosthetic group of citrate lyase

Schneider, K.; Bott, M.; Dimroth, B. M.

Journal Article

PreJuSER-26938

Biosynthesis of the prosthetic group of citrate lyase

Schneider, K. ; Dimroth, B. M. ; Bott, M.FZJ*

2000
American Chemical Society Columbus, Ohio

Biochemistry 39, 9438 - 9450 (2000)

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Please use a persistent id in citations: http://hdl.handle.net/2128/722

Abstract: 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.

Keyword(s): Acyl Carrier Protein: biosynthesis (MeSH) ; Amino Acid Sequence (MeSH) ; Apoproteins: biosynthesis (MeSH) ; Carbon-Sulfur Ligases: biosynthesis (MeSH) ; Carbon-Sulfur Ligases: genetics (MeSH) ; Coenzyme A: biosynthesis (MeSH) ; Enzyme Precursors: metabolism (MeSH) ; Escherichia coli: enzymology (MeSH) ; Escherichia coli: genetics (MeSH) ; Genes, Bacterial (MeSH) ; Molecular Sequence Data (MeSH) ; Multienzyme Complexes: biosynthesis (MeSH) ; Multienzyme Complexes: chemistry (MeSH) ; Multienzyme Complexes: genetics (MeSH) ; Multigene Family (MeSH) ; Operon (MeSH) ; Oxo-Acid-Lyases: biosynthesis (MeSH) ; Oxo-Acid-Lyases: chemistry (MeSH) ; Oxo-Acid-Lyases: genetics (MeSH) ; Structure-Activity Relationship (MeSH) ; Acyl Carrier Protein ; Apoproteins ; Enzyme Precursors ; Multienzyme Complexes ; dephosphocoenzyme A ; Coenzyme A ; Oxo-Acid-Lyases ; citrate (pro-3S)-lyase ; Carbon-Sulfur Ligases ; long-chain-fatty-acid-(acyl-carrier-protein) ligase ; citrate (pro-3S)-lyase ligase ; J