Douglas D. Randall

Cloning and molecular analyses of the Arabidopsis thaliana plastid pyruvate dehydrogenase subunits

Herein we report the first molecular description of the pyruvate dehydrogenase component of the higher plant plastid pyruvate dehydrogenase complex. The full-length cDNAs for the E1 alpha (1530 bp) and E1 beta (1441 bp) subunits of the Arabidopsis thaliana plastid pyruvate dehydrogenase contain open reading frames that encode polypeptides of 428 and 406 amino acids, respectively, with calculated molecular weight values of 47,120 and 44,208. The deduced amino acid sequences for Arabidopsis plastid E1 alpha and E1 beta have 61% and 68% identity to the odpA and odpB genes of the red alga Porphyra purpurea, respectively, but only 31% and 32% identity to the plant mitochondrial counterparts. Results of Southern analyses suggest that each subunit is encoded by a single gene. Northern blot analyses indicate expression of mRNAs of the appropriate size in Arabidopsis leaves.

Pyruvate dehydrogenase kinase from Arabidopsis thaliana: a protein histidine kinase that phosphorylates serine residues

Pyruvate dehydrogenase kinase (PDK) is the primary regulator of flux through the mitochondrial pyruvate dehydrogenase complex (PDC). Although PDKs inactivate mitochondrial PDC by phosphorylating specific Ser residues, the primary amino acid sequence indicates that they are more closely related to prokaryotic His kinases than to eukaryotic Ser/Thr kinases. Unlike Ser/Thr kinases, His kinases use a conserved His residue for phosphotransfer to Asp residues. To understand these unique kinases better, a presumptive PDK from Arabidopsis thaliana was heterologously expressed and purified for this investigation. Purified, recombinant A. thaliana PDK could inactivate kinase-depleted maize mitochondrial PDC by phosphorylating Ser residues. Additionally, A. thaliana PDK was capable of autophosphorylating Ser residues near its N-terminus, although this reaction is not part of the phosphotransfer pathway. To elucidate the mechanism involved, we performed site-directed mutagenesis of the canonical His residue likely to be involved in phosphotransfer. When His-121 was mutated to Ala or Gln, Ser-autophosphorylation was decreased by 50% and transphosphorylation of PDC was decreased concomitantly. We postulate that either (1) His-121 is not the sole phosphotransfer His residue or (2) mutagenesis of His-121 exposes an additional otherwise cryptic phosphotransfer His residue. Thus His-121 is one residue involved in kinase function.

Purification and properties of soybean nodule xanthine dehydrogenase

Abstract Xanthine dehydrogenase (EC 1.2.1.37), an essential enzyme for ureide metabolism was purified from the cytosol fraction of soybean nodules. The purified xanthine dehydrogenase was shown to be homogeneous by electrophoresis and a pI of 4.7 was determined by isoelectric focusing. The enzyme had a molecular weight of 285,000 and two subunits of molecular weight 141,000 each. The holoenzyme contained 1.7 (±0.7) mol Mo and 8.1 (±2.0) mol Fe/mol enzyme and the enzyme also contained FMN and is thus a molybdoironflavoprotein. Soybean xanthine dehydrogenase is the second enzyme in plants demonstrated to contain Mo and the first xanthine-oxidizing enzyme reported to contain FMN, rather than FAD as the flavin cofactor.

Purification and characterization of fructokinase from developing tomato (Lycopersicon esculentum Mill.) fruits

A procedure is described which allows the purification of fructokinase (EC 2.7.1.4) from young tomato fruit. The procedure yielded a 400-fold purification and two isoenzymes designated fructokinase I and II (FKI and FKII) were separated by anion-exchange chromatography. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the molecular mass was estimated to be 35 kDa. Gel filtration on Sepharose-12 indicated that for both fructokinases the functional form is a dimer. Two dimensional isoelectric focusing/SDS-PAGE combined with immunoblotting showed that FKI has two components with isoelectric points (pIs) of 6.42 and 6.55, while four components with pIs from 6.07 to 6.55 were detected for FKII. A mixture of both fructokinases showed that the components of FKI match the more alkaline components of FKII. The activity of both fructokinases increased with increasing pH to around 8.0 and equal activity was observed from 8.0 to 9.5. Both fructokinases were specific for fructose with Km values for fructose of 0.131 and 0.201 mM for FKI and FKII, respectively. At high concentrations (> 0.5 mM), fructose was also a strong inhibitor with inhibition constants (Ki) of 1.82 and 1.39 mM for FKI and FKII, respectively. The preferred phosphate donor for both isoforms was ATP, and Km values of 0.11 and 0.15 mM were observed for FKI and FKII. At low concentrations (0.05–0.2 mM), fructose exhibited noncompetitive inhibition with respect to ATP for both fructokinases. This inhibition pattern changed to uncompetitive when higher fructose concentrations (0.5–10 mM) were used. These data indicated that substrate addition is ordered, with ATP adding first. Inhibition by ADP was also affected by the fructose concentrations. At 0.5 mM fructose, FKI showed non-competitive inhibition by ADP with respect to ATP and this inhibition changed to uncompetitive when 3 mM fructose was used. The isoform FKII showed a competitive inhibition pattern for ADP at 0.5 mM fructose which also changed to uncompetitive when 3 mM fructose was used. The features of the regulation of both fructokinases suggest that this enzyme might have a relevant role in carbon metabolism during tomato fruit development.

Plant pyruvate dehydrogenase complex purification, characterization and regulation by metabolites and phosphorylation.

The pyruvate dehydrogenase complex was purified from mitochondria of cauliflower, Brassica oleracea var. botrytis floral buds to a specific activity of 5.4 mumol of NADH/min per mg of protein. The pyruvate dehydrogenase complex required CoASH, NAD+, thiamine pyrophosphate and Mg2+ for the oxidative decarboxylation of pyruvate. The kinetic analysis of the complex gave a series of parallel lines for all substrates. Product interaction patterns showed that NADH is competitive with NAD+; acetyl-CoA is competitive with CoASH; and NADH and acetyl-CoA uncompetitive with pyruvate. These kinetic patterns suggest a multisite ping-pong mechanism as described by Cleveland ((1973) J. Biol. Chem 248, 8353). The noncompetitive inhibition of NADH versus CoASH, and acetyl-CoASH versus NAD are not predicted by this mechanism. Regulation of the complex was more sensitive to the NADH/NAD+ ratio than acetyl-CoA/CoASH ratio. Hydroxypyruvate and glyoxylate inhibited the complex noncompetitively versus pyruvate. The pyruvate dehydrogenase complex was inactivated and phosphorylated by ATP. The ATP dependent inactivation is believed to be enzyme catalyzed by a pyruvate dehydrogenase complex kinase. However, no evidence was found for a plant pyruvate dehydrogenase complex phosphatase. The results suggest that the cauliflower pyruvate dehydrogenase complex is regulated by a phosphorylation-dephosphorylation mechanism.

The mitochondrial pyruvate dehydrogenase complex: nucleotide and deduced amino-acid sequences of a cDNA encoding the Arabidopsis thaliana E1 α-subunit

A cDNA encoding the E1 alpha subunit of the Arabidopsis thaliana (At) mitochondrial (mt) pyruvate dehydrogenase complex (PDC) was sequenced. The 1435-bp cDNA consists of a 1167-bp open reading frame encoding a 43.0-kDa polypeptide of 389 amino acids (aa) (pI 7.1). The plant E1 alpha subunit has 47-51% aa sequence identity with other eukaryotic sequences. Among the regions that are highly conserved are the aa surrounding phosphorylation sites 1 and 2 of the mammalian sequence, including the conserved Ser292 residue of At at site 1. An essential active site residue, Cys62 of the bovine subunit, is also conserved. A 32-aa presumptive mt targeting sequence is present at the N terminus.

Characterization of a monoclonal antibody recognizing the E1α subunit of plant mitochondrial pyruvate dehydrogenase

Summary We have isolated a monoclonal antibody that recognizes the E1α subunit of the plant mitochondrial pyruvate dehydrogenase complex. The antibody specifically recognizes the Ela subunit from maize seedling, pea leaf, and castor oil seed endosperm mitochondrial pyruvate dehydrogenases, but does not recognize the E1α subunit present in the plastid complexes from these plants. The pea mitochondrial pyruvate dehydrogenase complex was used for subsequent characterization of the antibody. Two-dimensional electrophoretic analysis of a phosphorylated pea mitochondrial pyruvate dehydrogenase complex preparation revealed that the monoclonal antibody recognizes all phosphorylated forms of the E1α subunit. Under these conditions, the only proteins recognized by the antibody are phosphorylated. Binding of the antibody to the pyruvate dehydrogenase complex inhibits both catalytic activity and phosphorylation of the E1α subunit, but does not significantly inhibit dephosphorylation. The monoclonal antibody recognizes mitochondrial E1α subunits from a variety of plant materials including monocot and dicot seedlings, and thermogenic and storage tissues. The antibody does not recognize the E1α subunit from rat liver or pig heart mitochondria, yeast, or bacteria. This highly specific antibody will be a useful tool for study of plant mitochondrial pyruvate dehydrogenase complexes.

Phosphorylation-dephosphorylation of pyruvate dehydrogenase complex from pea leaf mitochondria

The pyruvate dehydrogenase complex from pea leaf mitochondria was rapidly deactivated in the presence of 50 to 200 μm ATP. The deactivation of the complex requires Mg2+ as shown by EDTA inhibition of deactivation. Deactivation was inhibited by 0.1 to 1 mm pyruvate or dichloroacetate. Activation required 10 mM Mg2+ or Mn2+ but Ca2+ and K+ had no effect. Activation was inhibited by the phosphatase inhibitor, F−. Autoradiograms of nondissociating electrophoresis gel, crossed immunoelectrophoresis gels, and dissociating sodium dodecyl sulfate electrophoresis gels of the complex showed that one protein is labeled. Labeling of this protein is prevented by Mg2+, pyruvate, and dichloroacetate. The pyruvate dehydrogenase complex was isolated in a partially deactivated state and reactivation required exogenous Mg2+ and was inhibited by F−. These results are taken as conclusive evidence that the pyruvate dehydrogenase complex in pea leaf mitochondria undergoes interconversion between deactivated and activated states by covalent modification (phosphorylation-dephosphorylation) catalyzed by a kinase and phosphatase. Isolation of the complex in a partially deactivated (phosphorylated) state suggests a physiologically significant role for this regulatory mechanism.

Ureide biogenesis in leguminous plants

Abstract Allantion and allantoic acid are the predominant forms of organic nitrogen produced by N-fixing nodules of some important legume species. Only recently has the significance and biosynthetic origin of these substances in leguminous plants been realized.

Purification and characterization of pyruvate dehydrogenase complex from broccoli floral buds

Abstract The pyruvate dehydrogenase complex (PDC) was purified from Brassica oleracea var. italica floral buds to a specific activity of approximately 6 μmol of NADH formed/min/ mg of protein. The PDC had cofactor requirements for NAD + , thiamine pyrophosphate, coenzyme A, and a divalent cation (Mg 2+ , Ca 2+ , or Mn 2+ ). The enzyme catalyzed the oxidative decarboxylation of pyruvate at a rate threefold faster than 2-oxobutyrate but was inactive toward 2-oxoglutarate. The PDC was competively inhibited by acetyl-CoA against CoA and NADH against NAD + . The enzyme was shown to be more sensitive to regulation by NADH than acetyl-CoA.