Biosynthesis of the prosthetic group of citrate lyase

Biochemistry. 2000 Aug 8;39(31):9438-50. doi: 10.1021/bi000401r.


Citrate lyase (EC 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyl Carrier Protein / biosynthesis
  • Amino Acid Sequence
  • Apoproteins / biosynthesis
  • Carbon-Sulfur Ligases / biosynthesis
  • Carbon-Sulfur Ligases / genetics
  • Coenzyme A / biosynthesis
  • Enzyme Precursors / metabolism
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Genes, Bacterial
  • Molecular Sequence Data
  • Multienzyme Complexes / biosynthesis*
  • Multienzyme Complexes / chemistry
  • Multienzyme Complexes / genetics
  • Multigene Family
  • Operon
  • Oxo-Acid-Lyases / biosynthesis*
  • Oxo-Acid-Lyases / chemistry
  • Oxo-Acid-Lyases / genetics
  • Structure-Activity Relationship


  • Acyl Carrier Protein
  • Apoproteins
  • Enzyme Precursors
  • Multienzyme Complexes
  • dephosphocoenzyme A
  • Oxo-Acid-Lyases
  • citrate (pro-3S)-lyase
  • Carbon-Sulfur Ligases
  • long-chain-fatty-acid-(acyl-carrier-protein) ligase
  • citrate (pro-3S)-lyase ligase
  • Coenzyme A