Biosynthesis of the prosthetic group of citrate lyase.
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.
Schneider
K
K
Institut für Mikrobiologie, Eidgenössische Technische Hochschule Zürich, Switzerland.
Dimroth
P
P
Bott
M
M
D000213:Acyl Carrier Protein / Q000096:biosynthesis; D000595:Amino Acid Sequence; D001059:Apoproteins / Q000096:biosynthesis; D019730:Carbon-Sulfur Ligases / Q000096:biosynthesis / Q000235:genetics; D003065:Coenzyme A / Q000096:biosynthesis; D004792:Enzyme Precursors / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D005810:Multigene Family; D009876:Operon; D007652:Oxo-Acid-Lyases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship
D000213:Acyl Carrier Protein; D001059:Apoproteins; D004792:Enzyme Precursors; D009097:Multienzyme Complexes; C015787:dephosphocoenzyme A; D007652:Oxo-Acid-Lyases; C019909:citrate (pro-3S)-lyase; D019730:Carbon-Sulfur Ligases; C018749:long-chain-fatty-acid-(acyl-carrier-protein) ligase; C019908:citrate (pro-3S)-lyase ligase; D003065:Coenzyme A