Escherichia coli LipA Is a Lipoyl Synthase: In Vitro Biosynthesis of Lipoylated Pyruvate Dehydrogenase Complex from Octanoyl-Acyl Carrier Protein

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• 作者：J. Richard Miller ; Robert W. Busby ; Sean W. Jordan ; Jennifer Cheek ; Timothy F. Henshaw ; Gary W. Ashley ; Joan B. Broderick ; John E. Cronan ; Jr. ; and Michael A. Marletta
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：December 12, 2000
• 年：2000
• 卷：39
• 期：49
• 页码：15166 - 15178
• 全文大小：155K
• 年卷期：v.39,no.49(December 12, 2000)
• ISSN：1520-4995

文摘

The Escherichia coli lipA gene product has been genetically linked to carbon-sulfur bondformation 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 inE. coli as soluble proteins. The proteins were purified as a mixture of monomeric and dimeric speciesthat contain approximately four iron atoms per LipA polypeptide and a similar amount of acid-labilesulfide. Electron paramagnetic resonance and electronic absorbance spectroscopy indicate that the proteinscontain a mixture of [3Fe-4S] and [4Fe-4S] cluster states. Reduction with sodium dithionite results insmall quantities of an S = 1/2 [4Fe-4S]¹⁺ cluster with the majority of the protein containing a speciesconsistent with an S = 0 [4Fe-4S]²⁺ cluster. LipA was assayed for lipoate or lipoyl-ACP formation usingE. 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 withoctanoyl-ACP, LipB, apo-PDC, and S-adenosyl methionine (AdoMet), lipoylated PDC was formed. Asshown by this assay, octanoic acid is not a substrate for LipA. Confirmation that LipA catalyzes formationof lipoyl groups from octanoyl-ACP was obtained by MALDI mass spectrometry of a recombinant PDClipoyl-binding domain that had been lipoylated in a LipA reaction. These results provide informationabout the mechanism of LipA catalysis and place LipA within the family of iron-sulfur proteins thatutilize AdoMet for radical-based chemistry.