The role of loop and beta-turn residues as structural and functional determinants for the lipoyl domain from the Escherichia coli 2-oxoglutarate dehydrogenase complex

The lipoyl domain of the dihydrolipoyl succinyltransferase (E2o) component of the 2OGDH (2-oxoglutarate dehydrogenase) multienzyme complex houses the lipoic acid cofactor through covalent attachment to a specific lysine side chain residing at the tip of a beta-turn. Residues within the lipoyl-lysine beta-turn and a nearby prominent loop have been implicated as determinants of lipoyl domain structure and function. Protein engineering of the Escherichia coli E2o lipoyl domain (E2olip) revealed that removal of residues from the loop caused a major structural change in the protein, which rendered the domain incapable of reductive succinylation by 2-oxoglutarate decarboxylase (E1o) and reduced the lipoylation efficiency. Insertion of a new loop corresponding to that of the E. coli pyruvate dehydrogenase lipoyl domain (E2plip) restored lipoylation efficiency and the capacity to undergo reductive succinylation returned, albeit at a lower rate. Exchange of the E2olip loop sequence significantly improved the ability of the domain to be reductively acetylated by pyruvate decarboxylase (E1p), retaining approx. 10-fold more acetyl groups after 25 min than wild-type E2olip. Exchange of the beta-turn residue on the N-terminal side of the E2o lipoyl-lysine DK(A)/(V) motif to the equivalent residue in E2plip (T42G), both singly and in conjunction with the loop exchange, reduced the ability of the domain to be reductively succinylated, but led to an increased capacity to be reductively acetylated by the non-cognate E1p. The T42G mutation also slightly enhanced the lipoylation rate of the domain. The surface loop is important to the structural integrity of the protein and together with Thr42 plays an important role in specifying the interaction of the lipoyl domain with its partner E1o in the E. coli 2OGDH complex.     

Distinct modes of recognition of the lipoyl domain as substrate by the E1 and E3 components of the pyruvate dehydrogenase multienzyme complex

Two-dimensional (15)N-heteronuclear single-quantum coherence (HSQC) NMR studies with a di-domain (lipoyl domain+ linker+ peripheral subunit-binding domain) of the dihydrolipoyl acetyltransferase (E2) component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus allowed a molecular comparison of the need for lipoic acid to be covalently attached to the lipoyl domain in order to undergo reductive acetylation by the pyruvate decarboxylase (E1) component, in contrast with the ability of free lipoic acid to serve as substrate for the dihydrolipoyl dehydrogenase (E3) component. Tethering the lipoyl domain to the peripheral subunit-binding domain in a complex with E1 or E3 rendered the system more like the native enzyme complex, compared with the use of a free lipoyl domain, yet of a size still amenable to investigation by NMR spectroscopy. Recognition of the tethered lipoyl domain by E1 was found to be ensured by intensive interaction with the lipoyl-lysine-containing beta-turn and with residues in the protruding loop close to the beta-turn. The size and sequence of this loop varies significantly between species and dictates the lipoylated lipoyl domain as the true substrate for E1. In contrast, with E3 the main interaction sites on the tethered lipoyl domain were revealed as residues Asp41 and Ala43, which form a conserved sequence motif, DKA, around the lipoyl-lysine residue. No domain specificity is observed at this step and substrate channelling in the complex thus rests on the recognition of the lipoyl domain by the first enzyme, E1. The cofactor, thiamine diphosphate, and substrate, pyruvate, had distinct but contrasting effects on the E1/di-domain interaction, whereas NAD(+) and NADH had negligible effect on the E3/di-domain interaction. Tethering the lipoyl domain did not significantly change the nature of its interaction with E1 compared with a free lipoyl domain, indicative of the conformational freedom allowed by the linker in the movement of the lipoyl domain between active sites.     

Cross-talk between thiamin diphosphate binding and phosphorylation loop conformation in human branched-chain alpha-keto acid decarboxylase/dehydrogenase

The decarboxylase/dehydrogenase (E1b) component of the 4-megadalton human branched-chain alpha-keto acid dehydrogenase (BCKD) metabolic machine is a thiamin diphosphate (ThDP)-dependent enzyme with a heterotetrameric cofactor-binding fold. The E1b component catalyzes the decarboxylation of alpha-keto acids and the subsequent reductive acylation of the lipoic acid-bearing domain (LBD) from the 24-meric transacylase (E2b) core. In the present study, we show that the binding of cofactor ThDP to the E1b active site induces a disorder-to-order transition of the conserved phosphorylation loop carrying the two phosphorylation sites Ser(292)-alpha and Ser(302)-alpha, as deduced from the 1.80-1.85 A apoE1b and holoE1b structures. The induced loop conformation is essential for the recognition of lipoylated LBD to initiate E1b-catalyzed reductive acylation. Alterations of invariant Arg(287)-alpha, Asp(295)-alpha, Tyr(300)-alpha, and Arg(301)-alpha that form a hydrogen-bonding network in the phosphorylation loop result in the disordering of the loop conformation as elucidated by limited proteolysis, accompanied by the impaired binding and diminished reductive acylation of lipoylated LBD. In contrast, k(cat) values for E1b-catalyzed decarboxylation of the alpha-keto acid are higher in these E1b mutants than in wild-type E1b, with higher K(m) values for the substrate in the mutants. ThDP binding that orders the loop prevents phosphorylation of E1b by the BCKD kinase and averts the inactivation of wild-type E1b, but not the above mutants, by this covalent modification. Our results establish that the cross-talk between the bound ThDP and the phosphorylation loop conformation serves as a feed-forward switch for multiple reaction steps in the BCKD metabolic machine.     

A novel branched-chain amino acid metabolon. Protein-protein interactions in a supramolecular complex

The catabolic pathways of branched-chain amino acids have two common steps. The first step is deamination catalyzed by the vitamin B(6)-dependent branched-chain aminotransferase isozymes (BCATs) to produce branched-chain alpha-keto acids (BCKAs). The second step is oxidative decarboxylation of the BCKAs mediated by the branched-chain alpha-keto acid dehydrogenase enzyme complex (BCKD complex). The BCKD complex is organized around a cubic core consisting of 24 lipoate-bearing dihydrolipoyl transacylase (E2) subunits, associated with the branched-chain alpha-keto acid decarboxylase/dehydrogenase (E1), dihydrolipoamide dehydrogenase (E3), BCKD kinase, and BCKD phosphatase. In this study, we provide evidence that human mitochondrial BCAT (hBCATm) associates with the E1 decarboxylase component of the rat or human BCKD complex with a K(D) of 2.8 microM. NADH dissociates the complex. The E2 and E3 components do not interact with hBCATm. In the presence of hBCATm, k(cat) values for E1-catalyzed decarboxylation of the BCKAs are enhanced 12-fold. Mutations of hBCATm proteins in the catalytically important CXXC center or E1 proteins in the phosphorylation loop residues prevent complex formation, indicating that these regions are important for the interaction between hBCATm and E1. Our results provide evidence for substrate channeling between hBCATm and BCKD complex and formation of a metabolic unit (termed branched-chain amino acid metabolon) that can be influenced by the redox state in mitochondria.     

Heteronuclear NMR studies of the specificity of the post-translational modification of biotinyl domains by biotinyl protein ligase

The lipoyl domains of 2-oxo acid dehydrogenase multienzyme complexes and the biotinyl domains of biotin-dependent enzymes have homologous structures, but the target lysine residue in each domain is correctly selected for posttranslational modification by lipoyl protein ligase and biotinyl protein ligase, respectively. We have applied two-dimensional heteronuclear NMR spectroscopy to investigate the interaction between the apo form of the biotinyl domain of the biotin carboxyl carrier protein of acetyl-CoA carboxylase and the biotinyl protein ligase (BPL) from Escherichia coli. Heteronuclear multiple quantum coherence NMR spectra of the 15N-labelled biotinyl domain were recorded in the presence and absence of the ligase and backbone amide 1H and 15N chemical shifts were evaluated. Small, but significant, changes in chemical shift were found in two regions, including the tight beta-turn that houses the lysine residue targetted for biotinylation, and the beta-strand 2 and the loop that precedes it in the domain. When compared with the three-dimensional structure, sequence alignments of other biotinyl and lipoyl domains, and mutagenesis data, these results give a clear indication of how the biotinyl domain is both recognised by BPL and distinguished from the structurally related lipoyl domain to ensure correct posttranslational modification.     

Sequence conservation in the alpha and beta subunits of pyruvate dehydrogenase and its similarity to branched-chain alpha-keto acid dehydrogenase

Amino acid sequence comparison of 8 alpha and 6 beta subunits of the alpha-keto acid dehydrogenase (E1) component of the pyruvate dehydrogenase complex and branched-chain alpha-keto acid dehydrogenase complex form multiple species was performed by computer analysis. In addition to 2 previously recognized regions of homology in the alpha subunit, a 3rd region of extensive homology was identified in E1 alpha, and may be one of the sites involved in subunit interaction. E1 beta contains 4 regions of extensive homology. Region 1 contains 10 amino acids that are homologous to a 10-amino acid stretch in Escherichia coli E1. Regions 2 and 3 have sequence homologies with other dehydrogenases suggesting that these regions may be involved in catalysis.     

Solution Structure of the Lipoyl Domain of the 2-Oxoglutarate Dehydrogenase Complex fromAzotobacter vinelandii

The three-dimensional solution structure of the lipoyl domain of the 2-oxoglutarate dehydrogenase complex fromAzotobacter vinelandiihas been determined from nuclear magnetic resonance data by using distance geometry and dynamical simulated annealing refinement. The structure determination is based on a total of 580 experimentally derived distance constraints and 65 dihedral angle constraints. The solution structure is represented by an ensemble of 25 structures with an average root-mean-square deviation between the individual structures of the ensemble and the mean coordinates of 0.71 Å for backbone atoms and 1.08 Å for all heavy atoms. The overall fold of the lipoyl domain is that of a β-barrel-sandwich hybrid. It consists of two almost parallel four-stranded anti-parallel β-sheets formed around a well-defined hydrophobic core, with a central position of the single tryptophan 21. The lipoylation site, lysine 42, is found in a β-turn at the far end of one of the sheets, and is close in space to a solvent-exposed loop comprising residues 7 to 15. The lipoyl domain displays a remarkable internal symmetry that projects one β-sheet onto the other β-sheet after rotation of approximately 180° about a 2-fold rotational symmetry axis. There is close structural similarity between the structure of this 2-oxoglutarate dehydrogenase complex lipoyl domain and the structures of the lipoyl domains of pyruvate dehydrogenase complexes fromBacillus stearothermophilusandEscherichia coli, and conformational differences occur primarily in a solvent-exposed loop close in space to the lipoylation site. The lipoyl domain structure is discussed in relation to the process of molecular recognition of lipoyl domains by their parent 2-oxo acid dehydrogenase.     

Structure of the subunit binding domain and dynamics of the di-domain region from the core of human branched chain alpha-ketoacid dehydrogenase complex

The homo-24-meric dihydrolipoyl transacylase (E2) scaffold of the human branched-chain alpha-ketoacid dehydrogenase complex (BCKDC) contains the lipoyl-bearing domain (hbLBD), the subunit-binding domain (hbSBD) and the inner core domain that are linked to carry out E2 functions in substrate channeling and recognition. In this study, we employed NMR techniques to determine the structure of hbSBD and dynamics of several truncated constructs from the E2 component of the human BCKDC, including hbLBD (residues 1-84), hbSBD (residues 111-149), and a di-domain (hbDD) (residues 1-166) comprising hbLBD, hbSBD and the interdomain linker. The solution structure of hbSBD consists of two nearly parallel helices separated by a long loop, similar to the structures of the SBD isolated from other species, but it lacks the short 3(10) helix. The NMR results show that the structures of hbLBD and hbSBD in isolated forms are not altered by the presence of the interdomain linker in hbDD. The linker region is not entirely exposed to solvent, where amide resonances associated with approximately 50% of the residues are observable. However, the tethering of these two domains in hbDD significantly retards the overall rotational correlation times of hbLBD and hbSBD, changing from 5.54 ns and 5.73 ns in isolated forms to 8.37 ns and 8.85 ns in the linked hbDD, respectively. We conclude that the presence of the interdomain linker restricts the motional freedom of the hbSBD more significantly than hbLBD, and that the linker region likely exists as a soft rod rather than a flexible string in solution.     

Lipoic acid-dependent oxidative catabolism of alpha-keto acids in mitochondria provides evidence for branched-chain amino acid catabolism in Arabidopsis

Lipoic acid-dependent pathways of alpha-keto acid oxidation by mitochondria were investigated in pea (Pisum sativum), rice (Oryza sativa), and Arabidopsis. Proteins containing covalently bound lipoic acid were identified on isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis separations of mitochondrial proteins by the use of antibodies raised to this cofactor. All these proteins were identified by tandem mass spectrometry. Lipoic acid-containing acyltransferases from pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex were identified from all three species. In addition, acyltransferases from the branched-chain dehydrogenase complex were identified in both Arabidopsis and rice mitochondria. The substrate-dependent reduction of NAD(+) was analyzed by spectrophotometry using specific alpha-keto acids. Pyruvate- and alpha-ketoglutarate-dependent reactions were measured in all three species. Activity of the branched-chain dehydrogenase complex was only measurable in Arabidopsis mitochondria using substrates that represented the alpha-keto acids derived by deamination of branched-chain amino acids (Val [valine], leucine, and isoleucine). The rate of branched-chain amino acid- and alpha-keto acid-dependent oxygen consumption by intact Arabidopsis mitochondria was highest with Val and the Val-derived alpha-keto acid, alpha-ketoisovaleric acid. Sequencing of peptides derived from trypsination of Arabidopsis mitochondrial proteins revealed the presence of many of the enzymes required for the oxidation of all three branched-chain amino acids. The potential role of branched-chain amino acid catabolism as an oxidative phosphorylation energy source or as a detoxification pathway during plant stress is discussed.     

Solution structure of the lipoyl domain of the chimeric dihydrolipoyl dehydrogenase P64K from Neisseria meningitidis.

The antigenic P64K protein from the pathogenic bacterium Neisseria meningitidis is found in the outer membrane of the cell, and consists of two parts: an 81-residue N-terminal region and a 482-residue C-terminal region. The amino-acid sequence of the N-terminal region is homologous with the lipoyl domains of the dihydrolipoyl acyltransferase (E2) components, and that of the C-terminal region with the dihydrolipoyl dehydrogenase (E3) components, of 2-oxo acid dehydrogenase multienzyme complexes. The two parts are separated by a long linker region, similar to the linker regions in the E2 chains of 2-oxo acid dehydrogenase complexes, and it is likely this region is conformationally flexible. A subgene encoding the P64K lipoyl domain was created and over-expressed in Escherichia coli. The product was capable of post-translational modification by the lipoate protein ligase but not aberrant modification by the biotin protein ligase of E. coli. The solution structure of the apo-domain was determined by means of heteronuclear NMR spectroscopy and found to be a flattened beta barrel composed of two four-stranded antiparallel beta sheets. The lysine residue that becomes lipoylated is in an exposed beta turn that, from a [1H]-15N heteronuclear Overhauser effect experiment, appears to enjoy substantial local motion. This structure of a lipoyl domain derived from a dihydrolipoyl dehydrogenase resembles that of lipoyl domains normally found as part of the dihydrolipoyl acyltransferase component of 2-oxo acid dehydrogenase complexes and will assist in furthering the understanding of its function in a multienzyme complex and in the membrane-bound P64K protein itself.     

Branched-chain alpha-keto acid dehydrogenase complex from bovine kidney: radial distribution of mass determined from dark-field electron micrographs

Scanning transmission electron microscopy (STEM) was used to determine the radial distribution of mass within the bovine kidney branched-chain alpha-keto acid dehydrogenase complex (E1-E2) and its core enzyme, dihydrolipoamide acyltransferase (E2). The particle mass of E2 measured by STEM is (1.19 +/- 0.02) x 10(6). Assuming 24 subunits per E2 core, this value corresponds to a subunit molecular weight of (4.96 +/- 0.08) x 10(4), which agrees well with the subunit molecular weight estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 5.2 x 10(4) (Pettit et al., 1978) and that deduced from the gene sequence, 46,518 (Griffin et al., 1988). Thus, the STEM data reaffirms the 24-subunit model for this E2. Previous studies indicated that the E2 subunits contain an extended, outer lipoyl-bearing domain connected by a trypsin-sensitive segment to a compact, inner catalytic domain. The assemblage of 24 inner domains comprises a cubelike inner core. The quantity and spatial distribution of mass determined from STEM images for the E2 inner core are consistent with this model. The lipoyl-bearing domains are shown to occupy a zone defined by radii of 80-130 A over which the lipoyl moiety may range. This zone overlaps the positions of the 24 branched-chain alpha-keto acid dehydrogenase (E1) molecules, which apparently are located on the of the cubelike inner core.     

Inhibition of glycine oxidation by pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids in rat liver mitochondria: presence of interaction between the glycine cleavage system and alpha-keto acid dehydrogenase complexes

Pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids which were transaminated products of valine, leucine, and isoleucine inhibited glycine decarboxylation by rat liver mitochondria. However, glycine synthesis (the reverse reaction of glycine decarboxylation) was stimulated by those alpha-keto acids with the concomitant decarboxylation of alpha-keto acid added in the absence of NADH. Both the decarboxylation and the synthesis of glycine by mitochondrial extract were affected similarly by alpha-ketoglutarate and branched-chain alpha-keto acids in the absence of pyridine nucleotide, but not by pyruvate. This failure of pyruvate to have an effect was due to the lack of pyruvate oxidation activity in the mitochondrial extract employed. It indicated that those alpha-keto acids exerted their effects by providing reducing equivalents to the glycine cleavage system, possibly through lipoamide dehydrogenase, a component shared by the glycine cleavage system and alpha-keto acid dehydrogenase complexes. On the decarboxylation of pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids in intact mitochondria, those alpha-keto acids inhibited one another. In similar experiments with mitochondrial extract, decarboxylations of alpha-ketoglutarate and branched-chain alpha-keto acid were inhibited by branched-chain alpha-keto acid and alpha-ketoglutarate, respectively, but not by pyruvate. NADH was unlikely to account for the inhibition. We suggest that the lipoamide dehydrogenase component is an indistinguishable constituent among alpha-keto acid dehydrogenase complexes and the glycine cleavage system in mitochondria in nature, and that lipoamide dehydrogenase-mediated transfer of reducing equivalents might regulate alpha-keto acid oxidation as well as glycine oxidation.     

The C-terminal hinge region of lipoic acid-bearing domain of E2b is essential for domain interaction with branched-chain alpha-keto acid dehydrogenase kinase

The branched-chain alpha-keto acid dehydrogenase (BCKD) kinase (abbreviated as BCK) down-regulates activity of the mammalian mitochondrial BCKD complex by reversible phosphorylation of the decarboxylase (E1b) component of the complex. The binding of BCK to the holotransacylase (E2b) core of the BCKD complex results in the stimulation of BCK activity. Here we show that the lipoylated lipoic acid-bearing domain (lip-LBD) (residues 1-84) of E2b alone does not interact with BCK. However, lip-LBD constructs containing various lengths of the C-terminal hinge region of LBD are able to bind to BCK as measured by a newly developed solubility-based binding assay. Isothermal titration calorimetry measurements produced a dissociation constant of 8.06 x 10(-6) m and binding enthalpy of -3.68 kcal/mol for the interaction of BCK with a construct containing lip-LBD and the Glu-Glu-Asp-Xaa-Xaa-Glu sequence of the C-terminal hinge region of LBD. These thermodynamic parameters are similar to those obtained for binding of BCK to a lipoylated di-domain construct, which harbors LBD, the entire hinge region, and the downstream subunit-binding domain of E2b. Our data establish that the C-terminal hinge region of LBD containing the above negatively charged residues is essential for the interaction between the lip-LBD construct and BCK.     

Structural determinants of post-translational modification and catalytic specificity for the lipoyl domains of the pyruvate dehydrogenase multienzyme complex of Escherichia coli

The lipoyl domains of the dihydrolipoyl acyltransferase (E2p, E2o) components of the pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes are specifically recognised by their cognate 2-oxo acid decarboxylase (E1p, E1o). A prominent surface loop links the first and second beta-strands in all lipoyl domains, close in space to the lipoyl-lysine beta-turn. This loop was subjected to various modifications by directed mutagenesis of a sub-gene encoding a lipoyl domain of Escherichia coli E2p. Deletion of the loop (four residues) rendered the domain incapable of reductive acetylation by E. coli E1p in the presence of pyruvate, but insertion of a new loop (six residues) corresponding to that in the E2o lipoyl domain partly restored this ability, albeit with a much lower rate. However, the modified domain remained unable to undergo reductive succinylation by E1o in the presence of 2-oxoglutarate. Additional exchange of the two residues on the C-terminal side of the loop (V14A, E15T) had no effect. Insertion of a different four-residue loop also restored a limited ability to undergo reductive acetylation, but still significantly less than that of the wild-type domain. Exchanging the residue on the N-terminal side of the lipoyl-lysine beta-turn in the E2p and E2o domains (G39T), both singly and in conjunction with the loop exchange, had no effect on the ability of the E2p domain to be reductively acetylated but did confer a slight increase in susceptibility to reductive succinylation. All mutant E2p domains, apart from that with the loop deletion (LD), were readily lipoylated in vitro by E. coli lipoate protein ligase A; the E2p LD mutant could be lipoylated only at a significantly lower rate. Likewise, this domain exhibited 1D and 2D NMR spectra characteristic of a partially folded protein, whereas the spectra of mutants with modified loops were similar to those of the wild-type domain. The surface loop is evidently important to the structural integrity of the domain and may help to stabilize the thioester bond linking the acyl group to the reduced lipoyl-lysine swinging arm as part of the catalytic mechanism. Recognition of the lipoyl domain by its partner E1 appears to be a complex process and not attributable to any single determinant on the domain.     

Protein-protein interaction revealed by NMR T(2) relaxation experiments: the lipoyl domain and E1 component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

T(2) relaxation experiments in combination with chemical shift and site-directed mutagenesis data were used to identify sites involved in weak but specific protein-protein interactions in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. The pyruvate decarboxylase component, a heterotetramer E1(alpha(2)beta(2)), is responsible for the first committed and irreversible catalytic step. The accompanying reductive acetylation of the lipoyl group attached to the dihydrolipoyl acetyltransferase (E2) component involves weak, transient but specific interactions between E1 and the lipoyl domain of the E2 polypeptide chain. The interactions between the free lipoyl domain (9 kDa) and free E1alpha (41 kDa), E1beta (35 kDa) and intact E1alpha(2)beta(2) (152 kDa) components, all the products of genes or sub-genes over-expressed in Escherichia coli, were investigated using heteronuclear 2D NMR spectroscopy. The experiments were conducted with uniformly (15)N-labeled lipoyl domain and unlabeled E1 components. Major contact points on the lipoyl domain were identified from changes in the backbone (15)N spin-spin relaxation time in the presence and absence of E1(alpha(2)beta(2)) or its individual E1alpha or E1beta components. Although the E1alpha subunit houses the sequence motif associated with the essential cofactor, thiamin diphosphate, recognition of the lipoyl domain was distributed over sites in both E1alpha and E1beta. A single point mutation (N40A) on the lipoyl domain significantly reduces its ability to be reductively acetylated by the cognate E1. None the less, the N40A mutant domain appears to interact with E1 similarly to the wild-type domain. This suggests that the lipoyl group of the N40A lipoyl domain is not being presented to E1 in the correct orientation, owing perhaps to slight perturbations in the lipoyl domain structure, especially in the lipoyl-lysine beta-turn region, as indicated by chemical shift data. Interaction with E1 and subsequent reductive acetylation are not necessarily coupled.     

Enzymatic determination of the branched-chain alpha-keto acids

A spectrophotometric endpoint assay for determination of branched-chain alpha-keto acids is described. The assay depends on measurement of the NADH produced after addition of branched-chain alpha-keto acid dehydrogenase. Interference by pyruvate and alpha-ketobutyrate was eliminated by pretreating the sample with pyruvate dehydrogenase. The method yielded a peripheral venous plasma value of 59 +/- 5 microM (mean +/- SE) for the branched-chain alpha-keto acids of five overnight fasted healthy humans.     

alpha-Ketoglutarate dehydrogenase complex of Escherichia coli. A hybrid complex containing pyruvate dehydrogenase subunits from pyruvate dehydrogenase complex

The alpha-ketoglutarate dehydrogenase complex of Escherichia coli utilizes pyruvate as a poor substrate, with an activity of 0.082 units/mg of protein compared with 22 units/mg of protein for alpha-ketoglutarate. Pyruvate fully reduces the FAD in the complex and both alpha-keto[5-14C]glutarate and [2-14C]pyruvate fully [14C] acylate the lipoyl groups with approximately 10 nmol of 14C/mg of protein, corresponding to 24 lipoyl groups. NADH-dependent succinylation by [4-14C]succinyl-CoA also labels the enzyme with approximately 10 nmol of 14C/mg of protein. Therefore, pyruvate is a true substrate. However, the pyruvate and alpha-ketoglutarate activities exhibit different thiamin pyrophosphate dependencies. Moreover, 3-fluoropyruvate inhibits the pyruvate activity of the complex without affecting the alpha-ketoglutarate activity, and 2-oxo-3-fluoroglutarate inhibits the alpha-ketoglutarate activity without affecting the pyruvate activity. 3-Fluoro[1,2-14C]pyruvate labels about 10% of the E1 components (alpha-ketoacid dehydrogenases). The dihydrolipoyl transsuccinylase-dihydrolipoyl dehydrogenase subcomplex (E2E3) is activated as a pyruvate dehydrogenase complex by addition of E. coli pyruvate dehydrogenase, the E1 component of the pyruvate dehydrogenase complex. All evidence indicates that the alpha-ketoglutarate dehydrogenase complex purified from E. coli is a hybrid complex containing pyruvate dehydrogenase (approximately 10%) and alpha-ketoglutarate dehydrogenase (approximately 90%) as its E1 components.     

Structure and selectivity in post-translational modification: attaching the biotinyl-lysine and lipoyl-lysine swinging arms in multifunctional enzymes.

The post-translational attachment of biotin and lipoic acid to specific lysine residues displayed in protruding beta-turns in homologous biotinyl and lipoyl domains of their parent enzymes is catalysed by two different ligases. We have expressed in Escherichia coli a sub-gene encoding the biotinyl domain of E.coli acetyl-CoA carboxylase, and by a series of mutations converted the protein from the target for biotinylation to one for lipoylation, in vivo and in vitro. The biotinylating enzyme, biotinyl protein ligase (BPL), and the lipoylating enzyme, LplA, exhibited major differences in the recognition process. LplA accepted the highly conserved MKM motif that houses the target lysine residue in the biotinyl domain beta-turn, but was responsive to structural cues in the flanking beta-strands. BPL was much less sensitive to changes in these beta-strands, but could not biotinylate a lysine residue placed in the DKA motif characteristic of the lipoyl domain beta-turn. The presence of a further protruding thumb between the beta2 and beta3 strands in the wild-type biotinyl domain, which has no counterpart in the lipoyl domain, is sufficient to prevent aberrant lipoylation in E.coli. The structural basis of this discrimination contrasts with other forms of post-translational modification, where the sequence motif surrounding the target residue can be the principal determinant.     

Reductive acetylation of tandemly repeated lipoyl domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli is random order

In vitro deletion and site-directed mutagenesis of the aceF gene of Escherichia coli was used to generate dihydrolipoamide acetyltransferase (E2p) polypeptide chains containing various permutations and combinations of functional and non-functional lipoyl domains. A lipoyl domain was rendered non-functional by converting the lipoylatable lysine residue to glutamine. Two- and three-lipoyl domain E2p chains, with lipoyl-lysine (Lys244) substituted by glutamine in the innermost lipoyl domains (designated +/- and +/+/-, respectively), and similar chains with lipoyl-lysine (Lys143) substituted by glutamine in the outer lipoyl domains (designated -/+ and -/-/+), were constructed. In all instances, pyruvate dehydrogenase complexes were assembled in vivo around E2p cores composed of the modified peptide chains. All the complexes were essentially fully active in catalysis, although the complex containing the -/-/+ version of the E2p polypeptide chain showed a 50% reduction in specific catalytic activity. Similarly, active-site coupling in the complexes containing the +/-, +/+/- and -/+ constructions of the E2p chains was not significantly different from that achieved by the wild-type complex. However, active-site coupling in the complex containing the -/-/+ version of the E2p chain was slightly impaired, consistent with the reduced overall complex activity. These results indicate that during oxidative decarboxylation there is no mandatory order of reductive acetylation of repeated lipoyl domains within E2p polypeptide chains, and strongly suggest that the three tandemly repeated lipoyl domains in the wild-type E2p chain function independently in the pyruvate dehydrogenase complex.