Nucleotide sequence of the phosphoenolpyruvate carboxylase gene of the cyanobacterium Anacystis nidulans

Nucleotide sequence of the open reading frame (ORF) for the phosphoenolpyruvate carboxylase gene (ppc) of the cyanobacterium Anacystis nidulans was determined. The ORF consists of 3159 bp and codes for 1053 amino acid (aa) residues. The codon usage of the ppc of A. nidulans is not so markedly different from that of the Escherichia coli ppc, yet, in A. nidulans the preferred codons are AAG for lysine and CCC for proline, whereas those are seldom used in the E. coli ppc.     

Phosphoenolpyruvate carboxylase of Thermus sp.: role of a divergent glycine-rich region

In the phosphoenolpyruvate carboxylases (PEPCs) of mesophiles, a glycine(Gly)-rich region is well conserved and involved in catalytic activity, whereas the sequence of the corresponding region is considerably divergent in PEPC of Thermus sp. In this study, the Gly-rich region of Thermus PEPC was converted to that of Escherichia coli PEPC. The resulting mutant enzyme was as heat stable as the wild-type. However, its optimum temperature was markedly decreased. Thus, the divergent Gly-rich region of Thermus PEPC contributes to its catalytic activity but not to stability at high temperature.     

A fish oil diet rich in eicosapentaenoic acid reduces cyclooxygenase metabolites, and suppresses lupus in MRL-lpr mice

Dietary supplementation of fish oil as the exclusive source of lipid suppresses autoimmune lupus in MRL-lpr mice. This marine oil diet decreases the lymphoid hyperplasia regulated by the lpr gene, prevents an increase in macrophage surface Ia expression, reduces the formation of circulating retroviral gp70 immune complexes, delays the onset of renal disease, and prolongs survival. We show that a fatty acid component uniquely present in fish oil but not in vegetable oil decreases the quantity of dienoic prostaglandin E, thromboxane B, and prostacyclin normally synthesized by multiple tissues, including kidney, lung, and macrophages, and promotes the synthesis of small amounts of trienoic prostaglandin in autoimmune mice. We suggest that this change in endogenous cyclooxygenase metabolite synthesis directly suppresses immunologic and/or inflammatory mediators of murine lupus.     

Phosphoenolpyruvate carboxylase of Escherichia coli. Specificity of some compounds as activators at the site for fructose 1,6-bisphosphate, one of the allosteric effectors

An investigation was performed to elucidate some unusual phenomena which had been observed with phosphoenolpyruvate (PEP) carboxylase [EC 4.1.1.31] of Escherichia coli. (i) Fructose 1,6-bisphosphate (Fru-1,6-P2) and GTP--the allosteric activators--were competitive with each other in the activation. (ii) Some analogs of PEP such as DL-2-phospholactate and 2-phosphoglycolate, which behaved as inhibitors in the presence of the activator (acetyl-CoA or dioxane), activated the enzyme to some extent in the absence of the activator. (iii) Ammonium sulfate deprived the enzyme of sensitivity to Fru-1,6-P2 or GTP but had no effect on the sensitivity to other effectors. It was found that the activation by the analogs was lost upon desensitization of the enzyme to Fru-1,6-P2 by reaction with 2,4,6-trinitrobenzene sulfonate. The activation by the analogs was not observed in the presence of 200 mM ammonium sulfate. In the presence of lower concentrations (0.1 mM) of PEP, ammonium sulfate activated the enzyme at concentrations less than 700 mM but had an inhibitory effect on the desensitized enzyme. These findings suggest that the unusual phenomena described above are a result of binding of the phosphate esters and sulfate ions with the Fru-1,6-P2 site of the enzyme or the active site depending on the reaction conditions.     

Phosphorylation of phosphoenolpyruvate carboxylase is not essential for high photosynthetic rates in the C4 species Flaveria bidentis

Phosphoenolpyruvate carboxylase (PEPC; EC4.1.1.31) plays a key role during C(4) photosynthesis. The enzyme is activated by metabolites such as glucose-6-phosphate and inhibited by malate. This metabolite sensitivity is modulated by the reversible phosphorylation of a conserved serine residue near the N terminus in response to light. The phosphorylation of PEPC is modulated by a protein kinase specific to PEPC (PEPC-PK). To explore the role PEPC-PK plays in the regulation of C(4) photosynthetic CO(2) fixation, we have transformed Flaveria bidentis (a C(4) dicot) with antisense or RNA interference constructs targeted at the mRNA of this PEPC-PK. We generated several independent transgenic lines where PEPC is not phosphorylated in the light, demonstrating that this PEPC-PK is essential for the phosphorylation of PEPC in vivo. Malate sensitivity of PEPC extracted from these transgenic lines in the light was similar to the malate sensitivity of PEPC extracted from darkened wild-type leaves but greater than the malate sensitivity observed in PEPC extracted from wild-type leaves in the light, confirming the link between PEPC phosphorylation and the degree of malate inhibition. There were, however, no differences in the CO(2) and light response of CO(2) assimilation rates between wild-type plants and transgenic plants with low PEPC phosphorylation, showing that phosphorylation of PEPC in the light is not essential for efficient C(4) photosynthesis for plants grown under standard glasshouse conditions. This raises the intriguing question of what role this complexly regulated reversible phosphorylation of PEPC plays in C(4) photosynthesis.     

Thioredoxin-mediated reductive activation of a protein kinase for the regulatory phosphorylation of C4-form phosphoenolpyruvate carboxylase from maize.

The activity of phosphoenolpyruvate carboxylase (PEPC, EC4.1.1.31) for the C4 photosynthesis is known to be regulated mainly in response to light/dark transitions through reversible phosphorylation by a specific protein kinase (PK). PEPC-PK with an M(r) of 30 kDa was purified about 1.4 million-fold to homogeneity from maize leaves and characterized. The purified PEPC-PK was readily inactivated under mild oxidative conditions, but the activity could be recovered by dithiothreitol (DTT). The recovery by DTT was strongly accelerated by thioredoxin (Trx) from E. coli. Trxs of plant origin such as Trx-m from spinach chloroplast and Trx-h from rice cytoplasm were also effective. These results suggest the possibility of PEPC-PK being redox-regulated via Trx in vivo.     

The primary structure of phosphoenolpyruvate carboxylase of Escherichia coli. Nucleotide sequence of the ppc gene and deduced amino acid sequence

The nucleotide sequence of the ppc gene, the structural gene for phosphoenolpyruvate carboxylase [EC 4.1.1.31], of Escherichia coli K-12 was determined. The gene codes for a polypeptide comprising 883 amino acid residues with a calculated molecular weight of 99,061. The amino acid sequence deduced from the nucleotide sequence was entirely consistent with the protein chemical data obtained with the purified enzyme, including the NH2- and COOH-terminal sequences and amino acid composition. The coding region is preceded by two putative ribosome binding sites, and is followed closely by a good representative of rho-independent terminator. The codon usage in the ppc gene suggests a moderate expression of the gene. The secondary structure of the enzyme was predicted from the deduced amino acid sequence.     

Protection of murine systemic lupus by the Ea transgene without expression of I-E heterodimers

A high-level expression of the Ea transgene encoding the MHC class II I-E alpha-chain is very effective in the protection from systemic lupus erythematosus (SLE) in mice. However, it has not been elucidated whether this protection results from the induction or increased expression of I-E heterodimers or from the generation of I-E alpha-chain-derived peptides displaying high affinity for I-A molecules, because previous studies were conducted in lupus-prone mice expressing I-E beta-chains. To address this question, we assessed the protective effect of the Ea transgene in lupus-prone BXSB mice bearing the H2(q) haplotype (i.e., unable to express I-E heterodimers because of a deficiency in I-E beta-chains). We observed that the Ea transgene expression resulted in a marked suppression of the development of SLE in H2(q) BXSB mice despite the absence of I-E expression. The observed protection was not associated with any detectable levels of T cell depletion and regulatory T cell expansion. Significantly, transgenic I-E alpha-chains were substantially expressed on the surface of B lymphocytes and dendritic cells, but not of macrophages, without apparent formation of mixed-isotype heterodimers with I-A beta-chains. Our results demonstrate for the first time that the Ea transgene is able to prevent the development of SLE without induction of I-E heterodimer expression, indicating a critical role of I-E alpha-chains, but not I-E heterodimers, in the Ea transgene-mediated protection from SLE. Taken together, our data favor a model of autoimmunity prevention based on competition for Ag presentation between I-E alpha-chain-derived peptides and autoantigens.