A novel regulatory mechanism based upon a dynamic core structure for the mitochondrial pyruvate dehydrogenase complex?

The Arabidopsis thaliana genome includes three genes for mitochondrial dihydrolipoamide acetyltransferase, the E2-component of the mitochondrial pyruvate dehydrogenase complex (PDC). Two genes encode E2-proteins with a single lipoyl domain, while the third has a two-lipoyl domain structure. Transcripts for each E2 protein were expressed in all plant organs. Each recombinant AtmtE2 can individually form an icosahedral PDC core structure, and results from bimolecular fluorescence complementation assays are consistent with formation of hetero-core structures from all permutations of the AtmtE2 proteins. We propose a unique regulatory mechanism involving dynamic formation of hetero-core complexes that include both mono- and di-lipoyl forms of AtmtE2.

A functional genomic analysis of Arabidopsis thaliana PP2C clade D.

In the reference dicot plant Arabidopsis thaliana, the PP2C family of P-protein phosphatases includes the products of 80 genes that have been separated into ten multi-protein clades plus six singletons. Clade D includes the products of nine genes distributed among three chromosomes (APD1, At3g12620; APD2, At3g17090; APD3, At3g51370; APD4, At3g55050; APD5, At4g33920; APD6, At4g38520; APD7, At5g02760; APD8, At5g06750; and APD9, At5g66080). As part of a functional genomics analysis of protein phosphorylation, we retrieved expression data from public databases and determined the subcellular protein localization of the members of clade D. While the nine proteins have been grouped together based upon primary sequence alignments, we observed no obvious common patterns in expression or localization. We found chimera with the GFP associated with the nucleus, plasma membrane, the endomembrane system, and mitochondria in transgenic plants.

Asp295 stabilizes the active-site loop structure of pyruvate dehydrogenase, facilitating phosphorylation of ser292 by pyruvate dehydrogenase-kinase.

We have developed an in vitro system for detailed analysis of reversible phosphorylation of the plant mitochondrial pyruvate dehydrogenase complex, comprising recombinant Arabidopsis thalianaα2ß2-heterotetrameric pyruvate dehydrogenase (E1) plus A. thaliana E1-kinase (AtPDK). Upon addition of MgATP, Ser292, which is located within the active-site loop structure of E1α, is phosphorylated. In addition to Ser292, Asp295 and Gly297 are highly conserved in the E1α active-site loop sequences. Mutation of Asp295 to Ala, Asn, or Leu greatly reduced phosphorylation of Ser292, while mutation of Gly297 had relatively little effect. Quantitative two-hybrid analysis was used to show that mutation of Asp295 did not substantially affect binding of AtPDK to E1α. When using pyruvate as a variable substrate, the Asp295 mutant proteins had modest changes in k(cat), K(m), and k(cat)/K(m) values. Therefore, we propose that Asp295 plays an important role in stabilizing the active-site loop structure, facilitating transfer of the γ-phosphate from ATP to the Ser residue at regulatory site one of E1α.

A quantitative mass spectrometry-based approach for identifying protein kinase clients and quantifying kinase activity.

The Homo sapiens and Arabidopsis thaliana genomes are believed to encode more than 500 and 1000 protein kinases, respectively. Despite this abundance, few bona fide kinase-client relationships have been described in detail. Here we describe a quantitative mass spectrometry (MS)-based approach for identifying kinase-client proteins. During method development, we used the dedicated kinase pyruvate dehydrogenase kinase (PDK) for the in vitro assays. As kinase substrate, we used synthetic peptide cocktails and, in the process, demonstrated that the assay is both sensitive and specific. The method is also useful for characterizing protein kinase-substrate kinetics once the peptide substrate is detected. Applying a label-free spectral counting method, the activity of PDK was determined using the peptide substrate YHGH(292)SMSDPGSTYR derived from the pyruvate dehydrogenase E1alpha subunit sequence. The utility of spectral counting was further validated by studying the negative effect of Met oxidation on peptide phosphorylation. We also measured the activity of the unrelated calcium-dependent protein kinase 3 (CPK3), demonstrating the utility of the method in protein kinase screening applications.

Analysis of the catalytic mechanism of pyruvate dehydrogenase kinase.

It has been proposed that "Glu238" within the N-box of pyruvate dehydrogenase kinase (PDK) is a base catalyst. The pH dependence of k(cat) of Arabidopsis thaliana PDK indicates that ionizable groups with pK values of 6.2 and 8.4 are necessary for catalysis, and the temperature dependence of these values suggests that the acidic pK is due to a carboxyl- or imidazole-group. The E238 and K241 mutants had elevated K(m,ATP) values. The acidic pK value of the E238A mutant was shifted to 5.5. The H233A, L234H, and L234A mutants had the same pK values as wild-type AtPDK, contrary to the previous proposal of a "Glu-polarizing" His. Instead, we suggest that the conserved Glu, Lys, and Asn residues of the N-box contribute to coordinating Mg2+ in a position critical for formation of the PDK-MgATP-substrate ternary complex.

Expression and assembly of Arabidopsis thaliana pyruvate dehydrogenase in insect cell cytoplasm.

A vector was constructed for expression of Arabidopsis thaliana mitochondrial pyruvate dehydrogenase (E1) in the cytoplasm of Trichoplusia ni cells. The construct pDDR101 comprises the mature-E1alpha coding sequence under control of the Polh promoter, plus the mature-E1beta coding sequence under control of the p10 promoter. The E1alpha sequence was engineered to include an N-terminal His-tag. When protein samples were subjected to immobilized metal ion affinity chromatography, the alpha- and beta-subunits co-eluted, indicating association. When the recombinant protein sample was analyzed further by gel permeation chromatography, it was demonstrated that a significant amount eluted at a size consistent with assembly into an alpha2beta2 heterotetramer. Recombinant E1 was able to decarboxylate [1-14C]pyruvate and was a substrate for in vitro phosphorylation by E1-kinase.

Regulation of pyruvate dehydrogenase complex activity in plant cells.

The pyruvate dehydrogenase complex (PDC) is subjected to multiple interacting levels of control in plant cells. The first level is subcellular compartmentation. Plant cells are unique in having two distinct, spatially separated forms of the PDC; mitochondrial (mtPDC) and plastidial (plPDC). The mtPDC is the site of carbon entry into the tricarboxylic acid cycle, while the plPDC provides acetyl-CoA and NADH for de novo fatty acid biosynthesis. The second level of regulation of PDC activity is the control of gene expression. The genes encoding the subunits of the mt- and plPDCs are expressed following developmental programs, and are additionally subject to physiological and environmental cues. Thirdly, both the mt- and plPDCs are sensitive to product inhibition, and, potentially, to metabolite effectors. Finally, the two different forms of the complex are regulated by distinct organelle-specific mechanisms. Activity of the mtPDC is regulated by reversible phosphorylation catalyzed by intrinsic kinase and phosphatase components. An additional level of sensitivity is provided by metabolite control of the kinase activity. The plPDC is not regulated by reversible phosphorylation. Instead, activity is controlled to a large extent by the physical environment that exists in the plastid stroma.

The complex fate of alpha-ketoacids.

Plant cells are unique in that they contain four species of alpha-ketoacid dehydrogenase complex: plastidial pyruvate dehydrogenase, mitochondrial pyruvate dehydrogenase, alpha-ketoglutarate (2-oxoglutarate) dehydrogenase, and branched-chain alpha-ketoacid dehydrogenase. All complexes include multiple copies of three components: an alpha-ketoacid dehydrogenase/decarboxylase, a dihydrolipoyl acyltransferase, and a dihydrolipoyl dehydrogenase. The mitochondrial pyruvate dehydrogenase complex additionally includes intrinsic regulatory protein-kinase and -phosphatase enzymes. The acyltransferases form the intricate geometric core structures of the complexes. Substrate channeling plus active-site coupling combine to greatly enhance the catalytic efficiency of these complexes. These alpha-ketoacid dehydrogenase complexes occupy key positions in intermediary metabolism, and a basic understanding of their properties is critical to genetic and metabolic engineering. The current status of knowledge of the biochemical, regulatory, structural, genomic, and evolutionary aspects of these fascinating multienzyme complexes are reviewed.

Histidine mutagenesis of Arabidopsis thaliana pyruvate dehydrogenase kinase.

Pyruvate dehydrogenase kinase (PDK) is the primary regulator of flux through the mitochondrial pyruvate dehydrogenase complex (PDC). Analysis of the primary amino-acid sequences of PDK from various sources reveals that these enzymes include the five domains characteristic of prokaryotic two-component His-kinases, despite the fact that PDK exclusively phosphorylates Ser residues in the E1alpha subunit of the PDC. This seeming contradiction might be resolved if the PDK-catalyzed reaction employed a phospho-His intermediate. The results from pH-stability studies of autophosphorylated Arabidopsis thaliana PDK did not provide any support for a phospho-His intermediate. Furthermore, site-directed mutagenesis of the two most likely phosphotransfer His residues (H121 and H168) did not abolish either PDK autophosphorylation or the ability to transphosphorylate E1alpha. Thus, PDK is a unique type of protein kinase having a His-kinase-like sequence but Ser-kinase activity.

Developmental expression of the mitochondrial pyruvate dehydrogenase complex in pea (Pisum sativum) seedlings.

In order to better understand control of the mitochondrial pyruvate dehydrogenase complex (PDC), total catalytic activity was determined during development of the primary leaves of pea (Pisum sativum L.) seedlings, as well as in each leaf pair of 21-day-old plants. Activity of the PDC in clarified homogenates was highest in the youngest organs and then dropped dramatically as the leaves matured and became photosynthetically competent. As leaves began to senesce, total PDC activity dropped to zero. Steady-state mRNA levels were determined using E1 and E3 cDNA probes. The overall pattern of transcript abundance matched the pattern observed for total PDC activity; transcript levels for E1alpha and E1beta approached zero during senescence. Levels of the E1alpha, E1beta, E2 and E3 subunits of the PDC were analyzed in the same samples, using specific antibodies. Quantitation of the immunoblotting results throughout this developmental series showed a pattern in parallel with that of catalytic activity and mRNA levels, although the relative changes in subunit protein levels were not as extreme as the changes in activity. The exception to the global pattern was that of the E3 subunit: lipoamide dehydrogenase. Expression of this enzyme was highest in mature, fully expanded leaves, which were active in photosynthesis and photorespiration, reflecting the additional role of E3 as a component of glycine decarboxylase.