The role of the Saccharomyces cerevisiae lipoate protein ligase homologue, Lip3, in lipoic acid synthesis

Abstract

The covalent attachment of lipoate to the lipoyl domains (LDs) of the central metabolism enzymes pyruvate dehydrogenase (PDH) and oxoglutarate dehydrogenase (OGDH) is essential for their activation and thus for respiratory growth in Saccharomyces cerevisiae. A third lipoate-dependent enzyme system, the glycine cleavage system (GCV), is required for utilization of glycine as a nitrogen source. Lipoate is synthesized by extraction of it precursor, octanoyl-acyl carrier protein (ACP) from the pool of fatty acid biosynthetic intermediates. Alternatively, lipoate is salvaged from previously modified proteins or from growth media by lipoate protein ligases (Lpls). The first Lpl to be characterized, LplA of Escherichia coli, catalyzes two partial reactions: activation of the acyl chain by formation of acyl-AMP, followed by transfer of the acyl chain to LDs. There is a surprising diversity within the Lpl family of enzymes, several of which catalyze reactions other than ligation reactions. For example, the Bacillus subtils Lpl homologue LipM is an octanoyltransferase that transfers the octanoyl moiety from octanoyl-ACP to GCV. Another B. subtils Lpl homologue, LipL, transfers octanoate from octanoyl-GCV to other LDs in an amidotransfer reaction. Study of eukaryotic Lpls has lagged behind studies of the bacterial enzymes. We report that the Lip3 Lpl homologue of the yeast S. cerevisiae has octanoyl-CoA: protein transferase activity and discuss implications of this activity on the physiological role of Lip3 in lipoate synthesis.

Assembly of Lipoic Acid on Its Cognate Enzymes: an Extraordinary and Essential Biosynthetic Pathway

“…since they form insoluble inclusion bodies when expressed in E. coli and yeast (123). Several mutants with mutations in lipoate biosynthesis were isolated in the Tzagoloff laboratory (124) as strains having pleiotropic effects on mitochondrial metabolism (all of the lipoyl metabolism proteins are encoded by the nuclear genome but localize to the mitochondria).…”

“…In more recent work, the protein encoded by LIP3 (Lip3) was found to allow slow growth of an E. coli strain that lacked both the LplA lipoate ligase and the LipB octanoyl transferase, but only in the presence of octanoate (lipoate did not support growth but was not toxic) (123). Lip3 expression also restored 2-oxoacid dehydrogenase activity and E2 subunit lipoylation.…”

“…Growth in the presence of octanoate required that the E. coli strain have an active acyl-CoA synthetase, and acyl-CoA synthetase overexpression stimulated growth. Hence, a substrate of Lip3 seemed likely to be octanoyl-CoA, but the growth could also be explained by octanoyl-adenylate released by the synthetase being utilized by Lip3 (123). This was tested in vitro, and Lip3 was found to weakly modify both an E. coli lipoyl domain and yeast H protein (encoded by the GCV3 gene), using octanoyl-CoA as acyl donor.…”

“…The parental wildtype strain grew well on ethanol medium, whereas the LIP2, LIP3, and GCV3 mutant strains transformed with the empty vector failed to grow on the ethanol medium, even when it was supplemented with octanoate. The expression of the E. coli LplA derivative allowed growth of all three yeast mutant strains, indicating that the inactive yeast 2-oxoacid dehydrogenase proteins had been activated by lipoate attachment (123). The growth of the GCV3 mutant strain showed that the H protein is not necessary for respiration but, rather, is required for lipoylation of the 2-oxoacid dehydrogenases.…”

Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions

“…This can occur through LplA catalyzed activation of octanoic acid and octanoylation of the LCD [4], or through the action of an octanoyltransferase, LipB, which catalyzes the transfer of an octanoyl group from octanoyl-ACP to the lysine residue of the LCD [5]. Variations in the transferase and/or ligase enzymes required for de novo lipoyl cofactor biosynthesis have been reported in other microbes, but all proceed via octanoylated LCDs [6][7][8]. Assembly of the octanoyl LCD is followed by insertion of sulfur atoms at C6 and C8 of the octanoyl chain [3].…”