Biosynthesis of the Prosthetic Group of Citrate Lyase
- 8 July 2000
- journal article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 39 (31), 9438-9450
- https://doi.org/10.1021/bi000401r
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.Keywords
This publication has 19 references indexed in Scilit:
- Cloning and molecular characterization of the citrate utilization citMCDEFGRP cluster of Leuconostoc paramesenteroidesFEMS Microbiology Letters, 1999
- In vitro binding of the response regulator CitB and of its carboxy-terminal domain to A+T-rich DNA target sequences in the control region of the divergent citC and citS operons of Klebsiella pneumoniaeJournal of Molecular Biology, 1997
- Malonate Decarboxylase of Klebsiella pneumoniae Catalyses the Turnover of Acetyl and Malonyl Thioester Residues on a Coenzyme‐A‐Like Prosthetic GroupEuropean Journal of Biochemistry, 1996
- Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genesJournal of Molecular Biology, 1986
- Amino-Acid Sequence of Citrate-Lyase Acyl-Carrier Protein from Klebsiella aerogenesEuropean Journal of Biochemistry, 1978
- Structure of the prosthetic groups of citrate lyase and citramalate lyaseFEBS Letters, 1977
- The Prosthetic Group of Citrate‐Lyase Acyl‐Carrier ProteinEuropean Journal of Biochemistry, 1976
- Evaluation of the Protein Components of Citrate Lyase from Klebsiella aerogenesEuropean Journal of Biochemistry, 1975
- The Acyl‐Carrier Protein of Citrate LyaseEuropean Journal of Biochemistry, 1973
- Acetyl‐CoA‐Dependent Cleavage of Citrate on Inactivated Citrate LyaseEuropean Journal of Biochemistry, 1973