% IMPORTANT: The following is UTF-8 encoded. This means that in the presence % of non-ASCII characters, it will not work with BibTeX 0.99 or older. % Instead, you should use an up-to-date BibTeX implementation like “bibtex8” or % “biber”. @ARTICLE{Schneider:26938, author = {Schneider, K. and Dimroth, B. M. and Bott, M.}, title = {{B}iosynthesis of the prosthetic group of citrate lyase}, journal = {Biochemistry}, volume = {39}, issn = {0006-2960}, address = {Columbus, Ohio}, publisher = {American Chemical Society}, reportid = {PreJuSER-26938}, pages = {9438 - 9450}, year = {2000}, note = {Record converted from VDB: 12.11.2012}, 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 = {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 (NLM Chemicals) / Apoproteins (NLM Chemicals) / Enzyme Precursors (NLM Chemicals) / Multienzyme Complexes (NLM Chemicals) / dephosphocoenzyme A (NLM Chemicals) / Coenzyme A (NLM Chemicals) / Oxo-Acid-Lyases (NLM Chemicals) / citrate (pro-3S)-lyase (NLM Chemicals) / Carbon-Sulfur Ligases (NLM Chemicals) / long-chain-fatty-acid-(acyl-carrier-protein) ligase (NLM Chemicals) / citrate (pro-3S)-lyase ligase (NLM Chemicals) / J (WoSType)}, cin = {IBT}, ddc = {570}, cid = {I:(DE-Juel1)VDB184}, pnm = {Entwicklung von Mikroorganismen für die Herstellung von Primärmetaboliten}, pid = {G:(DE-Juel1)FUEK91}, shelfmark = {Biochemistry $\&$ Molecular Biology}, typ = {PUB:(DE-HGF)16}, pubmed = {pmid:10924139}, UT = {WOS:000088593300042}, url = {https://juser.fz-juelich.de/record/26938}, }