title: Escherichia coli LipA is a lipoyl synthase: in vitro biosynthesis of lipoylated pyruvate dehydrogenase complex from octanoyl-acyl carrier protein.
abstract: The Escherichia coli lipA gene product has been genetically linked to carbon-sulfur bond formation in lipoic acid biosynthesis [Vanden Boom, T. J., Reed, K. E., and Cronan, J. E., Jr. (1991) J. Bacteriol. 173, 6411-6420], although in vitro lipoate biosynthesis with LipA has never been observed. In this study, the lipA gene and a hexahistidine tagged lipA construct (LipA-His) were overexpressed in E. coli as soluble proteins. The proteins were purified as a mixture of monomeric and dimeric species that contain approximately four iron atoms per LipA polypeptide and a similar amount of acid-labile sulfide. Electron paramagnetic resonance and electronic absorbance spectroscopy indicate that the proteins contain a mixture of [3Fe-4S] and [4Fe-4S] cluster states. Reduction with sodium dithionite results in small quantities of an S = 1/2 [4Fe-4S](1+) cluster with the majority of the protein containing a species consistent with an S = 0 [4Fe-4S](2+) cluster. LipA was assayed for lipoate or lipoyl-ACP formation using E. coli lipoate-protein ligase A (LplA) or lipoyl-[acyl-carrier-protein]-protein-N-lipoyltransferase (LipB), respectively, to lipoylate apo-pyruvate dehydrogenase complex (apo-PDC) [Jordan, S. W., and Cronan, J. E. (1997) Methods Enzymol. 279, 176-183]. When sodium dithionite-reduced LipA was incubated with octanoyl-ACP, LipB, apo-PDC, and S-adenosyl methionine (AdoMet), lipoylated PDC was formed. As shown by this assay, octanoic acid is not a substrate for LipA. Confirmation that LipA catalyzes formation of lipoyl groups from octanoyl-ACP was obtained by MALDI mass spectrometry of a recombinant PDC lipoyl-binding domain that had been lipoylated in a LipA reaction. These results provide information about the mechanism of LipA catalysis and place LipA within the family of iron-sulfur proteins that utilize AdoMet for radical-based chemistry.
authors: Howard Hughes Medical Institute, Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, USA.
authors: J R
authors: JR
authors: Miller
authors: R W
authors: RW
authors: Busby
authors: S W
authors: SW
authors: Jordan
authors: J
authors: J
authors: Cheek
authors: T F
authors: TF
authors: Henshaw
authors: G W
authors: GW
authors: Ashley
authors: J B
authors: JB
authors: Broderick
authors: J E
authors: JE
authors: Cronan
authors: M A
authors: MA
authors: Marletta
mesh_terms: D000213:Acyl Carrier Protein / Q000378:metabolism*; D000215:Acylation; D001426:Bacterial Proteins / Q000378:metabolism*; D003001:Cloning, Molecular; D004227:Dithionite; D004926:Escherichia coli / Q000201:enzymology; D007501:Iron / Q000032:analysis; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D008956:Models, Chemical; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism; D013455:Sulfur / Q000032:analysis; D008063:Thioctic Acid / Q000096:biosynthesis*
chemical_list: D000213:Acyl Carrier Protein; D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; C079999:LipA protein, Bacteria; D011768:Pyruvate Dehydrogenase Complex; C088618:lipase activator protein, Bacteria; D004227:Dithionite; D013455:Sulfur; D008063:Thioctic Acid; D012436:S-Adenosylmethionine; D007501:Iron