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 its 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 medium by lipoate protein ligases (Lpls). The first Lpl to be characterized, LplA of Escherichia coli, catalyses two partial reactions: activation of the acyl chain by formation of acyl-AMP, followed by transfer of the acyl chain to lipoyl domains (LDs). There is a surprising diversity within the Lpl family of enzymes, several of which catalyse reactions other than ligation reactions. For example, the Bacillus subtilis Lpl homologue LipM is an octanoyltransferase that transfers the octanoyl moiety from octanoyl-ACP to GCV. Another B. subtilis Lpl homologue, LipL, transfers octanoate from octanoyl-GCV to other LDs in an amido-transfer 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.

Keywords: lipoate protein ligase; lipoic acid; mitochondrial integrity; octanoyl-CoA; protein transferase; respiration; tricarboxylic acid cycle.

Figure 1

Diversity of lipoate attachment mechanisms. In the biosynthetic pathways (dashed box), octanoyl-ACP (1) is extracted from the pool of fatty acid biosynthesis intermediates by an octanoyltransferase (LipB in E. coli, LipM in B. subtilis, or Lip2 in S. cerevisae) which transfers the octanoyl moiety to the LDs (e.g., that of the glycine cleavage system; black box). This is followed by sulfur insertion by a lipoate synthase (designated LipA in prokaryotes and Lip5 in S. cerevisiae). The LipL amidotransferases transfer the lipoyl moiety from lipoyl-GCV (3) to pyruvate dehydrogenase (white box), 2-oxoglutarate dehydrogenase and the branched chain oxoacid dehydrogenase (not shown). The lipoate ligase salvage pathways make use of free lipoate (5). LplA from E. coli, LplJ from B. subtilus and the LplA/B complex from T. acidophilum form lipoyl-PDH (4) for example. In L. monocytogenes LplA1 specifically modifies GCV, and the role of LplA2 is unknown (not shown). Also shown in the figure are other sources for octanoyl-ACP from free octanoate (6) and octanoyl-CoA (7) of fatty acid β-oxidation. Lipoamidases produce free lipoate by cleaving the coenzyme from degraded proteins.

Figure 2

Lip3 complementation of the E. coli ΔlipB ΔlplA strain QC146. A: Growth curves of strain QC146 carrying the Lip3 expression plasmid, pFH23, on either unsupplemented GMM or GMM supplemented with lipoate, octanoate, or a mixture of acetate and succinate as indicated. B: Replacing the Lip3 active site lysine (K249) with arginine or alanine greatly reduces or entirely eliminates the ability of Lip3 to support growth of QC146 on octanoate-supplemented GMM.

Figure 3

Detection of active lipoylated PDH and OGDH complexes in the E. coli ΔlipB ΔlplA strain QC146 expressing Lip3. (A) PDH and OGDH were assayed in extracts of strain QC146 carrying either the empty vector (I) or a plasmid that expressed Lip3 under arabinose control (II – IV). Growth medium was supplemented with lipoate or octanoate, both lipoate and octanoate or left unsupplemented as indicated. The highest levels of PDH and OGDH activities were observed in extracts of strain QC146 carrying the Lip3 expression plasmid pFH23 when octanoate was present in the growth media (IV). Symbols: * this result is significant relative to that of condition II (p-value = 0.003), and condition III (p-value = 0.02). Ŧ this result is significant relative to that of condition II (p-value = 0.0001), and condition III (p-value = 0.0005). The differences in PDH and OGDH activities between conditions II and III are not significant (not shown) (B) Western blot analysis using anti-lipoyl LD antibody of extracts of the strain QC146 carrying pFH23 cultured in LB containing arabinose, acetate and succinate with either lipoate (lane 2), octanoate (lane 3) or neither (lane 1). Whereas lipoylated SucB (the LD of OGDH) was detected in all three extracts, lipoylated AceF (the LD of PDH) was detected only in cells grown the presence of octanoate (3).

Figure 4

A: Lip3 complementation of the E. coli ΔlipB ΔlplA strain QC146 on octanoate-supplemented GMM requires the presence of the fadD encoded acyl-CoA synthetase. Shown are GMM plates unsupplemented (left plate), supplemented with octanoate (central plate), or with succinate and acetate (right plate). The top sector of each plate contains strain QC146 carrying a plasmid which expresses Lip3 whereas in the bottom sector of each plate the same strain carried a fadD deletion. B: Overexpression of the FadK acyl-CoA synthetase amplifies Lip3 complementation of the E. coli ΔlipB ΔlplA strain QC146. Growth curves on octanoate-supplemented GMM of QC146 carrying either the empty vector, a Lip3 expression plasmid or plasmids expressing both Lip3 and FadK as indicated.

Figure 5

In vitro detection of Lip3 octanoyl transferase activity by an electrophoresis mobility gel shift assay. Left gel: Lip3 reaction with LD with octanoyl-CoA (lane 1) or with ATP, octanoate and MgCl₂ (lane 2). Right gel: Lip3 activity with Gcv3 and octanoyl-CoA (lane 5). Lanes 3 and 4 were loaded with octanoyl-LD and apo-Gcv3 standards, respectively. Lip3 was active with both LD and Gcv3 when octanoyl-CoA was provided (lanes 1 and 5, respectively).

Figure 6

Growth phenotypes of S. cerevisiae lip2, lip3 and gcv3 strains transformed with different plasmids. All strains were derivatives of BY4741 and were grown on YP glucose, YP ethanol or YP ethanol supplemented with octanoate. Plates were incubated at 30° for up to four days. Panel A: the mutant strains transformed with empty vector. Panel B: the mutant strains transformed with pFH56 expressing mitochondrially targeted E. coli LplA. Panel C: the mutant strains transformed with pFH57 expressing mitochondrially targeted E. coli LipB. Panel D: the lip2 and lip3 strains transformed with pFH58 expressing mitochondrially targeted B. subtilis LipL.

Figure 7

Proposed pathway for lipoate synthesis/ attachment in S. cerevisiae which accounts for the observed octanoyl-Gcv3 checkpoint. Left: Lip2 specifically modifies Gcv3 using octanoyl- ACP from mitochondrial FA biosynthesis. Right: When all Gcv3 is octanoylated, octanoyl-ACP accumulates. An octanoyl-ACP: CoA transferase transfers the octanoyl moiety to CoA, providing substrate for Lip3 to modify PDH and OGDH. Lip5 catalyzes the sulfur insertions on the octanoyl moiety once it is attached to an LD (not shown).