The Marletta Lab

University of California, Berkeley

 

Research

People

Publications

Contact Info

Home Page

Lab Links

 Lipoic Acid Biosynthesis

 
 

Lipoic acid is a widely occurring coenzyme found in most prokaryotic and eukaryotic microorganisms,1 as well as many plant and animal tissues.2 Its principle role is in energy metabolism where it is covalently attached, through the carboxylic acid, to lysine residues3 forming an essential lipoamide in the E2 subunits of the pyruvate dehydrogenase complex (PDH)4 and the a-ketoglutarate dehydrogenase complex (KGDH).5 These large multienzyme complexes contain three units: a specialty dehydrogenase (E1p and E1o), a dihydrolipoamide acetyltransferase (E2p and E2o), and a lipoamide dehydrogenase (E3). In the E2p and E2o subunits, the lipoyllysyl moiety forms a "swinging arm" approximately 14 Å in size which aides in the transfer of acyl groups between the various enzymes within the multienzyme complex.2 Lipoic acid is crucial to the activity of these multienzyme complexes which are responsible for the synthesis of acetyl CoA used in the citric acid cycle.

Despite the extensive information available about the lipoic acid-dependent dehydrogenases, little is known about the enzymes involved in the biosynthesis of this coenzyme. The 8-carbon unit in lipoic acid is derived from octanoic acid and must undergo two carbon-sulfur bond formations on the way to lipoic acid.6 This reaction is chemically intriguing as the carbon-sulfur bonds are formed at unactivated carbons, presumably through a radical mechanism. To date, only one E. coli gene, lipA, has been genetically linked to this process.

Overexpression of lipA in Escherichia coli results in production of a soluble 36 kDa protein that can be isolated in both dimeric and monomeric forms. Each contains 4 Fe and 4 S per monomeric unit. Qualitative spectroscopic analyses including Resonance Raman, EPR, and UV-visible spectroscopies have identified the iron-sulfur center in the dimeric protein as a 4Fe-4S cubane, fully coordinated by cysteines.9 In identifying the structure of the iron-sulfur center, we hope to draw mechanistic parallels with other enzymes that contain 4Fe-4S centers and employ radical mechanisms. Our research is focused on determining if the iron-sulfur center in LipA is catalytic, purely structural or in some way regulatory.

In collaboration with John Cronan's laboratory at the University of Illinois we have developed a sensitive and quanitative assay for lipoate production by LipA. This assay has shown that lipoyl groups are synthesized by LipA from octanoyl-acyl carrier protein. We are currently involved in understanding the chemical steps of this fascinating reaction.

References

  1. Busby, R.W., Schelvis, J.P.M., Yu, D.S., Babcock, G.T. and Marletta, M.A. (1999). Lipoic acid biosynthesis: LipA is an Iron-Sulfur Protein. J. Am. Chem. Soc. 121: 4706-4707. [full text-PDF]

    2.  
  2. Herbert, A.A. and Guest, J.R. (1975). Lipoic acid content of Escherichia coli and other microorganisms. Arch. Microbiol.106: 259-266.

    3.  
  3. Reed, L.J. and Hackert, M.L. (1990). Structure-function relationships in dihydrolipoamide acyltransferases. J. Biol. Chem. 265: 8971-8974.

    4.  
  4. Reed, L.J. (1966). Chemistry and function of lipoic acid, pp. 99-126. In M. Florkin and E. H. Stotz (ed.), Comprehensive Biochemistry, vol. 14. Elsevier Publishing Company, New York.

    5.  
  5. Guest, J.R. and Russell, G.C. (1992). Complexes and complexities of the citric acid cycle in Escherichia coli. Curr. Top. Cell. Regul. 33: 231-247.

    6.  
  6. Perham, R.N. (1991). Domains, motifs, and linkers in 2-oxo acid dehydrogenase multienzyme complexes: a paradigm in the design of a multifunctional protein. Biochemistry 30: 8501-8512.

    7.  
  7. Parry, R.J. (1983). Biosynthesis of some sulfur-containing natural products. Investigations of the mechanism of carbon-sulfur bond formation. Tetrahedron 39: 1215-1238.

    8.  
  8. Jordan, S.W. and Cronan, J.E. (1997). A new metabolic link: the acyl carrier protein of lipid synthesis donates lipoic acid to the pyruvate dehydrogenase complex in Escherichia coli and mitochondria. J. Biol. Chem. 272: 17903-17906.

    9.  
  9. Reed, K.E. and Cronan, J. E. Jr. (1993). Lipoic acid metabolism in Escherichia coli: sequencing and functional characterization of the lipA and lipB genes. J. Bacteriol. 175: 1325-1336.