Lipoic acid functions as a cofactor of multienzyme complexes

Lipoic acid is a covalently attached cofactor of KADHs and is essential for their cat- alytic activity. KADHs consist of multimers of three independent proteins, namely a substrate specific α-keto acid decarboxylase (E1), an acyltransferase (E2) and a dihy- drolipoamide dehydrogenase (E3). These multienzyme complexes can be up to 10 MDa in size. In eukaryotes three KADHs are present, the pyruvate dehydrogenase (PDH), the branched chainα-keto acid dehydrogenase (BCDH) and theα-ketoglutarate dehydroge- nase (KGDH) (Perham, 2000).

Generally, KADHs convert an α-keto acid, NAD+ and coenzyme A (CoA) to CO2,

NADH and acyl-CoA and the reaction mechanism is shown in Figure 1.5. The substrate specific E1 subunit contains thiamine pyrophosphate (TPP) as a cofactor, and catalyses the decarboxylation of a α-keto acid, generating CO2 and acyl-TPP bound to the pro-

tein. Subsequently, the acyl-group is transferred to the lipoamide-group of the E2 subunit, which is covalently attached to a specific lysine residue of the acyltransferase subunit. The lipoamide transfers the acyl-group from the E1 subunit to coenzyme A to form acyl-CoA. The last step in the reaction is the re-oxidation of dihydrolipoamide by the FAD contain- ing E3 subunit producing NADH (Reed and Hackert, 1990, Perham, 2000).

The E2 subunits form homo-trimers, which assemble to either 24mers in a cubic organ- isation or 60mers in a pentagonal dodecahedron organisation (Reed and Hackert, 1990). This high molecular mass oligomer can be considered as the core of the multienzyme complexes, and E1 and E3 subunits bind to this core. The E2 protein consists of three dis- tinct domains. The N-terminal part of the protein is the lipoyl-domain, which contains the signature lysine residue that is post-translationally modified by attachment of lipoic acid. This domain is followed by a subunit binding domain, which confers binding of the E1 and E3 subunits to the E2 core structure. Subsequent to this is a catalytic domain found

C O C O R C OH O C O R OH C O S R C Lip SH O S

R Lip SH HS CoAHS CoA

C O S R C CoA O S R CoA C OH TPP R C OH TPP R S Lip S S Lip S SH Lip SH SH Lip SH TPP

E1

E2

E3

Figure 1.5.: General reaction mechanism of KADHs

The thiamine pyrophosphate dependent decarboxylase subunit (E1; blue) catalyses the decarboxy- lation of anα-keto acid and the subsequent reductive acylation of the lipoyl-group bound to the acyltransferase (E2; red). The E2 subunit catalyses the transfer of the acyl-group to CoA. The third subunit, the dihydrolipoamide dehydrogenase (E3; green), re-oxidises the reduced lipoyl-group of the E2-subunit with NAD+_{as the final electron acceptor.}

Abbreviations: RCOCOOH, α-keto acid; TPP, thiamine pyrophosphate; RCOH-TPP, hydroxyacyl-thiamine pyrophosphate; RCO-S-LipSH, acyl-lipoamide; LipS2, lipoamide;

Lip(SH)2, dihydrolipoamide; CoASH, Co-enzyme A; RCO-CoA, acyl-CoA

at the C-terminus, which catalyses the transfer of the acyl-rest to coenzyme A producing acyl-CoA and dihydrolipoamide. The domains are separated by flexible linker regions, which are ∼20-30 amino acids long, and rich in the amino acids proline and alanine, giv- ing them flexibility (Perham, 1991). Thus, these flexible linkers allow the lipoyl-domain to function as a "swinging-arm", transporting reaction intermediates from the E1 to the E2 and E3 subunits (Aevarsson et al., 1999, Mooney et al., 2002). E2 subunits possess one to three lipoyl-domains depending on the complex and organism they occur in. In in vitro assays it was shown that the lipoyl-domains alone can be expressed and are recognised by the lipoic acid attaching enzymes, and thus become post-translationally lipoylated (Ali and Guest, 1990, Dardel et al., 1990, Quinn et al., 1993). Figure 1.6 displays a schematic diagram of the P. falciparum KADH-E2 subunits and the H-protein, which is part of the glycine cleavage system described below.

Figure 1.6.: Schematic diagram of P. falciparum H-protein and KADH-E2 subunits

This figure displays schematically the structure of P. falciparum H-protein (A), PDH-E2 (B), BCDH-E2 (C) and KGDH-E2 (D). The KADH-E2 subunits consist of three domains, the lipoyl domains at the N-terminus (yellow), a substrate binding domain (green) and the catalytic domain at the C-terminus (red). The PDH-E2 possesses two lipoyl domains (9 kDa and 16 kDa in size) whereas the BCDH-E2 and KGDH-E2 only possess one lipoyl domain each (13 kDa and 12 kDa). The H-protein only consists of lipoyl-domain.

The E1 proteins confer substrate specificity to the complexes and require TPP and Mg2+

as cofactors for their activity. The dihydrolipoamide dehydrogenase (E3) is a flavopro- tein, which is responsible for the re-oxidation of dihydrolipoamide. In most organisms, the dihydrolipoamide dehydrogenase is shared between the different multienzyme com- plexes (Bourguignon et al., 1996). However, some organisms contain KADH specific E3 subunits (Lutziger and Oliver, 2000).

The PDH catalyses the oxidative decarboxylation of pyruvate to acetyl-CoA. The enzyme complex is generally found in the mitochondrion where it links glycolysis with the TCA cycle. Plants possess an additional PDH enzyme complex, which is present in the plas- tid, where it provides acetyl-CoA and NADH for fatty acid biosynthesis (Mooney et al., 2002). The situation in apicomplexan parasites is unique since these parasites possess only one PDH, which is present in the apicoplast (Foth et al., 2005). Accordingly, they possess two organelle specific E3 proteins (McMillan et al., 2005).

BCDH is located to the mitochondrion where it is involved in the degradation of the branched-chain amino acids valine, leucine and isoleucine. First, the branched-chain amino acid transaminase catalyses the production of the branched chain α-keto acids. Valine is converted into α-ketoisovaleric acid, leucine into α-ketoisocaproic acid and isoleucine intoα-keto-β-methylvaleric acid. Theseα-keto acids are then oxidative decar- boxylated by BCDH generating isobutyryl-CoA, isovaleryl-CoA and α-methylbutyryl-

CoA, respectively. Eventually, the acyl-CoA products can be converted to acetyl-CoA and/or succinyl-CoA for usage in the TCA cyle (Anderson et al., 1998). Homologues of BCDH-E1 and BCDH-E2 were identified in P. falciparum, and it has been shown that this enzyme complex is present in the mitochondrion (Günther et al., 2005, McMillan et al., 2005).

The KGDH is also found in the mitochondrion where it catalyses the oxidative decarboxy- lation ofα-ketoglutarate to succinyl-CoA as an integral part of the TCA cycle. Succinyl- CoA is also used for the biosynthesis of haem, whereas NADH is fed into the respiratory chain via complex I (NADH dehydrogenase). P. falciparum also possesses a KGDH, which is located in the mitochondrion (Günther et al., 2005, McMillan et al., 2005).

1.7.2.2. Glycine cleavage system

The glycine cleavage system (GCV) represents another mitochondrial multienzyme com- plex, which is dependent on lipoic acid. It catalyses the oxidative decarboxylation and deamination of glycine, generating CO2, NH3, NADH and N5,N10-methylene tetrahydro-

folate (CH2-THF), and the reaction mechanism is shown in Figure 1.7. The GCV consists

of multiple copies of four protein subunits, namely P-protein, H-protein, T-protein and L-protein. The P-protein is a pyridoxal phosphate dependent decarboxylase, which catal- yses the decarboxylation of glycine and the reductive methylamination of the lipoamide, which is covalently attached to the H-protein. The T-protein requires THF for its activity, and catalyses the transfer of methylene to THF and the subsequent release of NH3. The

H-protein then reacts with the L-protein, the dihydrolipoamide dehydrogenase described above, to oxidise the dihydrolipoamide with NAD+ as the final electron acceptor. The released CH2-THF reacts with another glycine molecule, resulting in the formation of

serine, a reaction that is catalysed by serine hydroxymethyltransferase (SHMT), which is closely associated with the GCV (Douce et al., 2001).

Overall, the reaction mechanism of the GCV is very similar to the one observed in KADHs, with lipoic acid function as a shuttle transporting reaction intermediates to the different active sites in the complex. As the lipoyl-moieties in the KADH-E2 subunits, lipoic acid is covalently attached via an amide linkage to theε-amino group of a conserved lysine residue of the H-protein. Structurally, the H-protein is related to the lipoyl-domains of the KADH-E2 subunits and could be seen as the "lipoyl-domain" of the GCV (Figure 1.6).

Figure 1.7.: Reaction mechanism of the glycine cleavage system

The P-protein, a pyridoxal phosphate dependent decarboxylase, catalyses the decarboxylation of glycine and the subsequent transfer of the methylamine-group to the lipoamide-group of the H- protein. The methylamine-group is passed on the T-protein, which catalyses its deamination. The L-protein is responsible for the re-oxidization of the dihydrolipoamide of the H-protein with NAD+ _{as the final electron acceptor. A serine hydroxymethyltransferase associated with the}

glycine cleavage system catalyses the reaction of glycine with the methyl-group bound to tetrahy- drofolate to form serine. The image is taken from Douce et al. (2001) with permission from Elsevier.

Abbreviations: P, P-protein; H, H-protein; T, T-protein; L, L-protein, SHMT, serine hydrox- ymethyltransferase; THF, tetrahydrofolate; Hmet, methylaminated H-protein; Hred, reduced form

of H-protein; Hox, oxidized form of H-protein

A detailed structure of the GCV is not known, but the subunit stoichiometry of plant GCV was investigated and it was shown to contain four P-protein homo-dimers, 27 H- protein monomers, nine T-protein monomers and two L-protein homo-dimers, with the H-proteins forming the core of this complex (Oliver et al., 1990). Homologues of all sub- units, except for the P-protein, have been identified in the P. falciparum genome. Further analyses suggest that the GCV is present in the mitochondrion, since all identified sub- units possess potential mitochondrial transit peptides at their N-termini (Salcedo et al., 2005).