The remarkable structural and functional organization of the eukaryotic pyruvate dehydrogenase complexes

The three-dimensional reconstruction of the bovine kidney pyruvate dehydrogenase complex (M(r) approximately 7.8 x 10(6)) comprising about 22 molecules of pyruvate dehydrogenase (E(1)) and about 6 molecules of dihydrolipoamide dehydrogenase (E(3)) with its binding protein associated with the 60-subunit dihydrolipoamide acetyltransferase (E(2)) core provides considerable insight into the structural and functional organization of the largest multienzyme complex known. The structure shows that potentially 60 centers for acetyl-CoA synthesis are organized in sets of three at each of the 20 vertices of the pentagonal dodecahedral core. These centers consist of three E(1) molecules bound to one E(2) trimer adjacent to an E(3) molecule in each of 12 pentagonal openings. The E(1) components are anchored to the E(1)-binding domain of the E(2) subunits through an approximately 50-A-long linker. Three of these linkers emanate from the outside edges of the triangular base of the E(2) trimer and form a cage around its base that may shelter the lipoyl domains and the E(1) and E(2) active sites. The docking of the atomic structures of E(1) and the E(1) binding and lipoyl domains of E(2) in the electron microscopy map gives a good fit and indicates that the E(1) active site is approximately 95 A above the base of the trimer. We propose that the lipoyl domains and its tether (swinging arm) rotate about the E(1)-binding domain of E(2,) which is centrally located 45-50 A from the E(1), E(2), and E(3) active sites, and that the highly flexible breathing core augments the transfer of intermediates between active sites.     

Disorders of pyruvate carboxylase and the pyruvate dehydrogenase complex

Summary The most common defect associated with deficiency of the pyruvate dehydrogenase (PDH) complex occurs in the E₁ component, specifically due to mutations in the X-linked E₁ gene. Clinical sequelae of these mutations, which range from severe neonatal lactic acidosis to carbohydrate-sensitive ataxia, can be different in males and females depending on the nature of the mutation and, in the case of females, on the X-inactivation pattern in different tissues. Males have a high representation of missense mutations among the patient cohort, while females are much more likely to have DNA rearrangements, particularly toward the 3 end of the coding sequence of the gene. Missplicing mutations involving exon 6 deletion have been reported, as has a missense mutation conferring true thiamin-responsiveness of the enzyme and the patient's clinical symptoms.Pyruvate carboxylase deficiency, on the other hand, is a true autosomal recessive disease, though it has high occurrences in particular ethnic groups, especially in Algonkian-speaking Amerindians and in Arabs. In the former group the defect is a simple type in which material cross-reactive to pyruvate carboxylase antibody is present in cultured cells (CRM^+ve). In the latter group, cross-reacting material is rarely present (CRM^–ve). The CRM^+ve patients can survive into teenage years with careful supervision, while the CRM^–ve patients have complications due to hyperammonaemia and dysfunction of the urea cycle and rarely survive beyond 3 months of life.     

Disorders of the pyruvate dehydrogenase complex

Pyruvate dehydrogenase deficiency may be a non-specific consequence of many different neurological degenerative disorders. There are also serious methodological problems in estimating the activity of this enzyme complex.     

Source pool geometry and the assembly of continental avifaunas

Classical niche-assembly models propose that the composition of biotic communities in continental landscapes is determined chiefly by the autecology of species, interspecific competition, and the diversity of resources and habitats within a region. In contrast, stochastic models propose that simulation algorithms can replicate the macroecological patterns, if not the mechanisms, of community assembly. Despite fundamental differences in assumptions, both categories of models assume that species are drawn from regional source pools. We explored the implications of source pool geometry on the assembly of avian communities with an analysis of assemblage dispersion fields, which can be visualized by overlaying the geographic ranges of all species that occur in an assemblage. Contours of species richness surrounding focal quadrats illustrate the decay rate of assemblage similarity with distance and the probable geometry of assemblage source pools. We used a geographic database for 2,891 species of South American birds to characterize dispersion fields for assemblages sampled by 1 degrees latitude-longitude quadrats (n = 1,676). We show that the median range size of dispersion fields varies by an order of magnitude across the continent. Because abundance generally correlates with geographic range size within taxonomic groups, the number of individuals per species in avifaunal source pools must also vary by an order of magnitude. Most significantly, dispersion field geometry was surprisingly asymmetrical and exhibited complex geographical patterns that were associated with the distribution of biomes. These results are broadly consistent with the predictions of niche-assembly models but offer little support for stochastic assembly models.     

Reconstitution of the Escherichia coli pyruvate dehydrogenase complex.

The binding of pyruvate dehydrogenase and dihydrolipoyl dehydrogenase (flavoprotein) to dihydrolipoyl transacetylase, the core enzyme of the E. coli pyruvate dehydrogenase complex [EC 1.2.4.1:pyruvate:lipoate oxidoreductase (decaryboxylating and acceptor-acetylating)], has been studied using sedimentation equilibrium analysis and radioactive enzymes in conjunction with gel filtration chromatography. The results show that the transacetylase, which consists of 24 apparently identical polypeptide chains organized into a cube-like structure, has the potential to bind 24 pyruvate dehydrogenase dimers in the absence of flavoprotein and 24 flavoprotein dimers in the absence of pyruvate dehydrogenase. The results of reconstitution experiments, utilizing binding and activity measurements, indicate that the transacetylase can accommodate a total of only about 12 pyruvate dehydrogenase dimers and six flavoprotein dimers and that this stoichiometry, which is the same as that of the native pyruvate dehydrogenase complex, produces maximum activity. It appears that steric hindrance between the relatively bulky pyruvate dehydrogenase and flavoprotein molecules prevents the transacetylase from binding 24 molecules of each ligand. A structural model for the native and reconstituted pyruvate dehydrogenase complexes is proposed in which the 12 pyruvate dehydrogenase dimers are distributed symmetrically on the 12 edges of the transacetylase cube and the six flavoprotein dimers are distributed in the six faces of the cube.     

丙酮酸脱氢酶复合物E2亚单位的克隆表达与临床应用

目的 构建丙酮酸脱氢酶复合物的E2亚单位(PDC-E2)的表达载体。方法 采用逆转录聚合酶链反应技术从人淋巴细胞中扩增出PDC-E2的基因片段，克隆至pExsecI表达载体进行诱导表达，并对表达产物进行western blot和酶联免疫吸附法鉴定。结果 成功构建了表达载体pExSecI／PDC—E2，表达产物能特异性的被原发性胆汁性肝硬化(PBC)患者血清中的自身抗体识别。结论 获得PDC-E2蛋白的高效表达，为利用原核表达PDC-E2对PBC患者进行血清学检测奠定了基础。     

Role of the regulatory subunit of bovine pyruvate dehydrogenase phosphatase.

Bovine pyruvate dehydrogenase phosphatase (PDP) is a Mg2+-dependent and Ca2+-stimulated heterodimer that is a member of the protein phosphatase 2C family and is localized to mitochondria. Insight into the function of the regulatory subunit of PDP (PDPr) has been gained. It decreases the sensitivity of the catalytic subunit of PDP (PDPc) to Mg2+. The apparent Km of PDPc for Mg2+ is increased about 5-fold, from about 0.35 mM to 1.6 mM. The polyamine spermine increases the sensitivity of PDP but not PDPc to Mg2+, apparently by interacting with PDPr. PDPc but not PDP can use the phosphopeptide RRAT(P)VA as a substrate. These observations are interpreted to indicate that PDPr blocks or distorts the active site of PDPc and that spermine produces a conformational change in PDPr that reverses its inhibitory effect. These findings suggest that PDPr may be involved in the insulin-induced activation of the mitochondrial PDP in adipose tissue, which is characterized by a decrease in its apparent Km for Mg2+.     

Heterogeneous expression of protein and mRNA in pyruvate dehydrogenase deficiency.

Deficiency of pyruvate dehydrogenase [pyruvate:lipoamide 2-oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1], the first component of the pyruvate dehydrogenase complex, is associated with lactic acidosis and central nervous system dysfunction. Using both specific antibodies to pyruvate dehydrogenase and cDNAs coding for its two alpha and beta subunits, we characterized pyruvate dehydrogenase deficiency in 11 patients. Three different patterns were found on immunologic and RNA blot analyses. (i) Seven patients had immunologically detectable crossreactive material for the alpha and beta proteins of pyruvate dehydrogenase. (ii) Two patients had no detectable crossreactive protein for either the alpha or beta subunit but had normal amounts of mRNA for both alpha and beta subunits. (iii) The remaining two patients also had no detectable crossreactive protein but had diminished amounts of mRNA for the alpha subunit of pyruvate dehydrogenase only. These results indicate that loss of pyruvate dehydrogenase activity may be associated with either absent or catalytically inactive proteins, and in those cases in which this enzyme is absent, mRNA for one of the subunits may also be missing. When mRNA for one of the subunits is lacking, both protein subunits are absent, suggesting that a mutation affecting the expression of one of the subunit proteins causes the remaining uncomplexed subunit to be unstable. The results show that several different mutations account for the molecular heterogeneity of pyruvate dehydrogenase deficiency.     

Cloning and cDNA sequence of the dihydrolipoamide dehydrogenase component human alpha-ketoacid dehydrogenase complexes.

cDNA clones comprising the entire coding region for human dihydrolipoamide dehydrogenase (dihydrolipoamide:NAD+ oxidoreductase, EC 1.8.1.4) have been isolated from a human liver cDNA library. The cDNA sequence of the largest clone consisted of 2082 base pairs and contained a 1527-base open reading frame that encodes a precursor dihydrolipoamide dehydrogenase of 509 amino acid residues. The first 35-amino acid residues of the open reading frame probably correspond to a typical mitochondrial import leader sequence. The predicted amino acid sequence of the mature protein, starting at the residue number 36 of the open reading frame, is almost identical (greater than 98% homology) with the known partial amino acid sequence of the pig heart dihydrolipoamide dehydrogenase. The cDNA clone also contains a 3' untranslated region of 505 bases with an unusual polyadenylylation signal (TATAAA) and a short poly(A) track. By blot-hybridization analysis with the cDNA as probe, two mRNAs, 2.2 and 2.4 kilobases in size, have been detected in human tissues and fibroblasts, whereas only one mRNA (2.4 kilobases) was detected in rat tissues.     

Mechanism of action of the pyruvate dehydrogenase multienzyme complex from Escherichia coli

The extent of cooperativity among the polypeptide chain components in the overall reaction catalyzed by the pyruvate dehydrogenase multienzyme complex from Escherichia coli has been studied. Selective inactivation of the pyruvate dehydrogenase component with thiamin thiazolone pyrophosphate demonstrates that no cooperativity between this component and the overall catalytic reaction occurs: the amount of overall complex activity is directly proportional to the fraction of active pyruvate dehydrogenase component. The transacetylase component has two lipoic acid residues on each of its polypeptide chains that can be modified by N-[(3)H]ethylmaleimide in the presence of pyruvate and thiamin pyrophosphate. The kinetics of the loss of overall complex activity due to modification of the lipoyl residues on the transacetylase component by maleimide reagents shows that not all lipoic acids are coupled into the overall catalytic reaction and that acyl-group and electron pair transfer involving two or more lipoic acids per catalytic cycle must occur. Finally, full complex activity is found when only half the normal flavin content is present. The results indicate that extensive communication among lipoic acids in acyl-group and electron pair transfer must exist in the normal catalytic mechanism. These results are consistent with the average distances between catalytic sites measured by energy transfer experiments.     

Activity of the pyruvate dehydrogenase complex in the mammary gland of normal and diabetic rats

Summary After parturition there is a 10 fold increase in the actual and total activity of the PDH complex in the mammary gland, which can be explained by an increased amount of enzyme protein. There is a marked difference between the activity state of the PDH complex in the suckled and unsuckled gland of the same animals. In fasting rats the active form of the PDH complex is decreased. This effect is further enhanced by inhibition of suckling. In the diabetic state the PDH_a activity is reduced, but the change is statistically insignificant. The decreased milk production during diabetes results from the reduction of the total mass of the gland. The total activity of the PDH complex is the same in fetal and neonatal liver of the rat. Whereas the PDH complex is fully activated before parturition, there is a significant decrease in the active form of the pyruvate dehydrogenase complex in the liver of newborn rats.Supported by the Landesamt für Forschung des Landes Nordrhein-Westfalen. A part of these data have been presented on the Joint Meeting of the Biochemical Societies of Belgium, the Federal Republic of Germany and the Netherlands, 1974 [7].     

细粒棘球绦虫丙酮酸脱氢酶的重组表达及生物信息学分析

目的 目的 克隆表达细粒棘球绦虫丙酮酸脱氢酶 （EgPDH） 基因, 并对其进行生物信息学预测与分析。 方法 方法 提取细粒棘球绦虫Total?RNA并反转录成cDNA, 以此为模板扩增目的基因。将目的基因连接至pET28b构建重组质粒并转化大肠杆菌BL21 （DE3） 进行重组表达。采用SignalP4.1、 TMHMM sever v.2.0和TargetP1.1对EgPDH编码蛋白序列分别进行信号肽、 跨膜区及亚细胞定位的预测。采用SMART分析EgPDH编码蛋白结构域, 用BLASTP和GeneDoc进行EgPDH同源序列比对及保守位点分析, 并采用MEGA6软件邻接法构建系统进化树。 结果 结果 成功克隆并构建重组质粒pET28b? EgPDH, 目的基因大小约1 080 bp, 重组蛋白以可溶性形式表达。EgPDH为信号肽的分泌蛋白, 并含转酮酶结构域, 其高度保守酶活性位点分别为Glu57 、 Leu72 、 Ile86 、 Phe114 。系统进化树分析显示EgPDH与多房棘球绦虫PDH亲缘关系最近。 结 结论 论 成功克隆表达了细粒棘球绦虫EgPDH基因并进行了生物信息学预测分析, 为进一步研究该蛋白功能奠定了基础。     

Scaffold proteins and assembly of multiprotein signaling complexes

Intracellular signaling involves assembly and regulation of multiprotein complexes. These complexes are functional units of signal transduction and are a means by which protein networks carry out tasks within the cell. One mechanism to influence the components, the subcellular localization, and the activity of these complexes, involves scaffold proteins. Scaffold proteins facilitate signal transduction by tethering molecules together and serving as molecular backbones for signaling complex assembly. Recent studies, particularly in the field of signaling kinases, have considerably advanced our understanding of the role that scaffold proteins play within multiprotein complexes in cardiac and other cell types.     

Biochemical nature of pyruvate dehydrogenase complex in the patient with primary lactic acidaemia

Summary The biochemical nature of the pyruvate dehydrogenase complex (PDHC) in muscle was studied in a patient with pyruvate dehydrogenase complex deficiency. The enzyme activity was approximately 30% of the control level and the apparentK _m value of the enzyme was similar to the control value. The immunoblot pattern of each subunit protein, E₁, E₁, the component X, E₂ and E₃, was comparable to that of the control on both one-and two-dimensional electrophoresis, the staining of each subunit protein being reduced in intensity, corresponding to the reduced enzyme activity. The enzyme deficiency is likely to be quantitative rather than qualitative, although the actual mechanism is unknown.     

T-cell responses to the components of pyruvate dehydrogenase complex in primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is an autoimmune condition that results in destruction of the intrahepatic biliary epithelial cells and is characterized by autoantibodies to pyruvate dehydrogenase complex (PDC). The portal tract T-cell infiltrate and up-regulation of HLA class I, HLA class II, and cell adhesion molecules such as intercellular adhesion molecule-1 on the biliary epithelial cells suggest that T cells play a significant role in mediating this damage. The authors have characterized the peripheral blood T-cell proliferative responses of 24 PBC patients and 48 controls (20 normal, 28 non-PBC chronic liver disease) to the dominant autoantigen PDC, and its constituent components E1, E2 and protein X (which co-purify), and E3. A significant proportion of both PBC patients and controls showed T-cell responses to whole PDC (12 of 24 vs. 24 of 48 SI > 2.5 P = NS) and E1 (15 of 24 vs. 25 of 48 P = NS). Responses to PDC and El are thus seen in normal individuals and are not limited to PBC patients. T-cell responses to E2/X were seen in most PBC patients (14 of 24), but in only a small number of controls (6 of 48, P < .0001), responses to E2/X being significantly more frequent in pre-cirrhotic PBC patients (stages I to III, 12 of 15) than cirrhotic (stage IV, 2 of 9 P < .05). Peripheral blood T-cell responses to E2/X are thus strongly associated with early PBC. Responses to E3 were low in both PBC patients and controls. No differences were seen in responses to the control antigen tetanus toxoid between PBC patients and controls. These in vitro observations are compatible with the view that peripheral mechanisms may play a significant role in maintaining self-tolerance to PDC in the normal state, and that the expression of specific Tcell responses to PDC-E2/X in vivo in PBC patients may be a consequence of impairment of these mechanisms of peripheral tolerance.