Purification and properties of the chloroplastic form of biotin holocarboxylase synthetase from Arabidopsis thaliana overexpressed in Escherichia coli

Holocarboxylase synthetases (HCSs) are key enzymes in biotin utilisation in both prokaryotes and eukaryotes. In a previous study, we demonstrated that, in plants, HCS activity is localised in cytosol, chloroplasts and mitochondria. We also described the cloning and sequencing of a full-length cDNA encoding an Arabidopsis thaliana HCS isoform with a putative organelle-transit peptide. In the study reported here, this cDNA was used to construct an overproducing Escherichia coli strain. The recombinant enzyme was isolated using an efficient three-step purification procedure. Polyclonal antibodies raised against pure HCS were produced to elucidate the subcellular localisation of this protein. Immunodetection carried out by Western blotting of isolated pea leaf subcellular compartments specifically revealed a single polypeptide that was ascribed to the chloroplast compartment. Immunocytochemistry of thin-cut sections from tobacco leaves, transformed by the complete coding sequence of A. thaliana HCS cDNA via Agrobacterium tumefaciens, confirmed that the enzyme encoded by this cDNA is the chloroplastic isoform. Moreover, physicochemical, biochemical and kinetic properties of the pure recombinant HCS were determined. The native recombinant enzyme is a 37-kDa monomer. In contrast to the major part of HCS activity measured in leaf extracts, the recombinant chloroplastic enzyme did not require addition of Mg2+ to be fully active, but was substantially inhibited by EDTA. This suggested that the chloroplastic HCS may contain a tightly-bound divalent cation required for enzyme activity. The recombinant enzyme was able to biotinylate efficiently apo-biotin carboxyl carrier protein (BCCP) from E. coli and apo-methylcrotonoyl-CoA carboxylase (MCCase) from A. thaliana. Apparent Km values for the enzyme substrates D-biotin, ATP and apo-MCCase were found to be 130 nM, 4.4 microM and 32 microM, respectively. Steady-state kinetic analyses of the HCS-catalysed reaction were investigated with respect to reaction mechanism and inhibition by AMP, one of the end-products of the enzyme-catalysed reaction. Substrate interaction and product inhibition patterns indicated that ATP and D-biotin bind sequentially, in an ordered manner, to the enzyme and that ATP or D-biotin and apo-BCCP bind in ping-pong fashion.     

Inflorescence-specific genes from Arabidopsis thaliana encoding glycine-rich proteins

Genomic and cDNA clones for three inflorescence-specific genes from Arabidopsis thaliana were isolated and characterized. The genes are tandemly organized in the genome on a 10 kb fragment. The expression of these genes is coordinately regulated in a developmental and organ-specific pattern. They are expressed predominantly in anthers at the later stage of flower development. The primary structure of the encoded gene products exhibits comparable features consisting of a hydrophobic domain at the N-terminal region followed by repeated glycine-rich motifs. Little homology is observed either between the glycine-rich domain of the three genes or with previously described glycine-rich proteins from other plant species.     

cDNA sequence and overexpression of chloroplast chaperonin 21 from Arabidopsis thaliana

Higher plant chloroplasts contain a 21-kDa protein, chaperonin 21 (Cpn21), that is a functional homolog of the chaperonin 10 (Cpn10). The chloroplast Cpn21 polypeptide consists of two Cpn10-like domains fused together in tandem. We describe here the cDNA sequence of the Cpn21 (AtCpn21) precursor protein from Arabidopsis thaliana. The deduced amino acid sequence of the AtCpn21 precursor protein, 253 amino acids long, shows 61% identity with the spinach Cpn21 protein. The AtCpn21 precursor protein contains the typical chloroplast transit peptide of 51 amino acids at its aminoterminus and the two Cpn10-like domains which exhibits 46% sequence identity to each other. The predicted mature-sized polypeptide of AtCpn21 was expressed in Escherichia coli as a soluble 21-kDa protein. Gel-filtration and chemical cross-linking analyses showed that the recombinant mature AtCpn21 protein forms a stable homo-oligomer composed of three or four polypeptides.     

Identification and characterization of a novel cap-binding protein from Arabidopsis thaliana

Cap-binding proteins specifically bind to the 7-methyl guanosine (m7G) functional group at the 5' end of eukaryotic mRNAs. A novel Arabidopsis thaliana protein has been identified that has sequence similarity to cap-binding proteins but is clearly a different form of the protein. The most obvious primary sequence difference is the substitution of two of the eight conserved tryptophan residues with other aromatic amino acids in the novel protein. Analogous forms of this novel protein appear to be present in other higher eukaryotes but not in yeast. Analysis of the native and recombinant forms of the novel protein by retention on m7GTP-Sepharose indicate that it is a functional cap-binding protein. Measurements of the dissociation constant for this protein indicate that it binds m7GTP 5-20-fold tighter than eukaryotic initiation factor (eIF)(iso)4E. The novel protein also supports the initiation of translation of capped mRNA in vitro. Biochemical analysis and yeast two-hybrid data indicate that it interacts with eIF(iso)4G to form a complex. Based on these observations, this protein appears to be able to function as a cap-binding protein and is given the designation of novel cap-binding protein (nCBP).     

Cloning of the cDNA for glutamyl-tRNA synthetase from Arabidopsis thaliana

High-sensitivity titration calorimetry is used to measure changes in enthalpy, heat capacity and protonation for the binding of captopril to the angiotensin I-converting enzyme (ACE; EC 3.4.15.1). The affinity of ACE to captopril is high and changes slightly with the pH, because the number of protons linked to binding is low. The determination of the enthalpy change at different pH values suggests that the protonated group in the captopril-ACE complex exhibits a heat protonation of approximately -30 kJ/mol. This value agrees with the protonation of an imidazole group. The residues which may become protonated in the complex could be two histidines existing in two active sites, which are joined to the amino acids coordinated to Zn2+. Calorimetric measurements indicate that captopril binds to two sites in the monomer of ACE, this binding being enthalpically unfavorable and being dominated by a large positive entropy change. Thus, binding is favored by both electrostatic and hydrophobic interactions. The temperature dependence of the free energy of binding deltaG degrees is weak because of the enthalpy-entropy compensation caused by a large heat capacity change, deltaCp =-4.3+/-0.1 kJ/K/mol of monomeric ACE. The strong favorable binding entropy and the negative deltaCp indicate both a large contribution to binding due to hydrophobic effects, which seem to originate from dehydration of the ligand-protein interface, and slight conformational changes in the vicinity of the active sites.     

A probarley lectin processing enzyme purified from Arabidopsis thaliana seeds

An aspartic proteinase was purified from the seeds of Arabidopsis thaliana (ecotype RLD) using affinity chromatography on pepstatin-agarose and ion exchange chromatography. The purified enzyme is optimally active at pH 3.5 and completely inhibited by pepstatin A. The purified Arabidopsis aspartic proteinase contains four subunits (apparent molecular weights 31, 28.5, 15 and 6 kDa), two of which are probably linked by disulfide bridges. These properties are similar to the aspartic proteinase previously isolated from barley seeds. The amino acid sequence of the peptide subunits corresponds exactly with the sequence of the previously isolated cDNA for the Arabidopsis aspartic proteinase. The Arabidopsis enzyme processed probarley lectin in vitro at the carboxy-terminus between phenylalanine and alanine, the same place where the barley enzyme cleaves the lectin in vitro. The aspartic proteinase appears to be the major enzyme processing the lectin in seeds as pepstatin A inhibited this activity in a crude seed extract.     

Purification and immunological analysis of phospholipase D from castor bean endosperm

Phospholipase D (EC 3.1.4.4) has been implicated in diverse cellular processes, but its physiological role is not well established in plants. In order to develop immunological and molecular biology approaches to address the problem, we report here the immunological analysis and N-terminal amino acid sequence of a cytosolic phospholipase D from castor bean (Ricinus communis L.). The enzyme was purified to apparent homogeneity from germinating castor bean endosperm. The specific activity of the purified enzyme was enhanced by approximately 670-fold with an overall yield of 4%. Its molecular mass was estimated at 92 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of this enzyme was KLVENIEETVGFGKG. Polyclonal antibodies were raised against the purified enzyme. The antibodies inhibited the activity of transphosphatidylation more than that of hydrolysis of phospholipase D. The differential effect on the two activities of this enzyme implies that different active sites on this enzyme may be involved in the two reactions. Immunoblot analyses showed that the amounts of phospholipase D protein relative to the total endosperm proteins increased during the first 5 days of germination. The antibodies cross-reacted to proteins from several tested plant species, and those proteins had molecular masses similar to that of castor bean phospholipase D. These results indicate that the expression of phospholipase D in castor bean changes according to growth stages and that phospholipase D enzymes of different plant species are structurally related.     

Polyamines and paraquat toxicity in Arabidopsis thaliana

The morbidly obese have a disproportionately greater risk of hypertension, diabetes, and coronary artery disease than their lean or less seriously obese counterparts. Roux-en-Y gastric bypass surgery has been found to be highly effective in inducing, and sustaining, weight loss in individuals with morbid obesity. The purpose of the present study was to examine the effects of weight loss with Roux-en-Y gastric bypass surgery (GBP) on blood pressure, fasting blood glucose, and the lipid/lipoprotein status of 61 morbidly obese women and 21 men. Anthropometric and blood pressure assessments and blood samples for glucose and lipid/lipoprotein analyses were obtained before surgery and at 6 to 12 months postoperatively. By this time, morbidly obese (MO) males and females had lost 33% and 30% of their initial body weight, respectively, along with significant reductions in fasting blood glucose (p < 0.01) and systemic blood pressure (p < 0.05). Weight loss with GBP was also associated with significant reductions in the apoprotein B-containing lipoproteins and the triglyceride and cholesterol composition of these particles. There was a trend (p < 0.10) toward increased serum levels of high density lipoprotein (HDL)-cholesterol following GBP, and significant (p < 0.05) improvement in HDL subfraction distribution and composition. These findings demonstrate the effectiveness of GBP in inducing metabolic changes in the MO population, which may reduce the risk of coronary artery disease, diabetes, and hypertension.     

An allelic series of blue fluorescent trp1 mutants of Arabidopsis thaliana

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CYP86A1 from Arabidopsis thaliana encodes a cytochrome P450-dependent fatty acid omega-hydroxylase

The A. thaliana EST database was screened using consensus motifs derived from P450 families CYP52 and CYP4 catalyzing the omega-hydroxylation of fatty acids and alkanes in Candida and in mammals. One EST cDNA fragment was detected in this way and the corresponding full-length cDNA was cloned from a cDNA library of A. thaliana. This cDNA coded the first member of a new plant P450 family and was termed CYP86A1. The deduced peptide sequence showed highest homology with P450s from families 4 and 52. To confirm the catalytic function, CYP86A1 was expressed in a yeast overexpressing its own NADPH-P450 reductase. Efficient expression was evidenced by spectrophotometry, SDS-PAGE and catalytic activity. CYP86A1 was found to catalyze the omega-hydroxylation of saturated and unsaturated fatty acids with chain lengths from C12 to C18 but not of hexadecane. Genomic organization analyzed by Southern blot suggested a single gene encoding CYP86A1 in A. thaliana.     

Diversity and molecular evolution of the RPS2 resistance gene in Arabidopsis thaliana

The RPS2 gene in Arabidopsis thaliana governs resistance to strains of the bacterial pathogen, Pseudomonas syringae pv. tomato, that express the avrRpt2 gene. The two loci are involved in a gene-for-gene interaction. Seventeen accessions of A. thaliana were sequenced to explore the diversity present in the coding region of the RPS2 locus. An unusually high level of nucleotide polymorphisms was found (1.26%), with nearly half of the observed polymorphisms resulting in amino acid changes in the RPS2 protein. Seven haplotypes (alleles) were identified and their evolutionary relationships deduced. Several of the alleles conferring resistance were found to be closely related, whereas susceptibility to disease was conferred by widely divergent alleles. The possibility of selection at the RPS2 locus is discussed.     

UV- and gamma-radiation sensitive mutants of Arabidopsis thaliana

Arabidopsis seedlings repair UV-induced DNA damage via light-dependent and independent pathways. The mechanism of the "dark repair" pathway is still unknown. To determine the number of genes required for dark repair and to investigate the substrate-specificity of this process we isolated mutants with enhanced sensitivity to UV radiation in the absence of photoreactivating light. Seven independently derived UV sensitive mutants were isolated from an EMS-mutagenized population. These fell into six complementation groups, two of which (UVR1 and UVH1) have previously been defined. Four of these mutants are defective in the dark repair of UV-induced pyrimidine [6-4]pyrimidinone dimers. These four mutant lines are sensitive to the growth-inhibitory effects of gamma radiation, suggesting that this repair pathway is also involved in the repair of some type of gamma-induced DNA damage product. The requirement for the coordinate action of several different gene products for effective repair of pyrimidine dimers, as well as the nonspecific nature of the repair activity, is consistent with nucleotide excision repair mechanisms previously described in Saccharomyces cerevisiae and nonplant higher eukaryotes and inconsistent with substrate-specific base excision repair mechanisms found in some bacteria, bacteriophage, and fungi.     

Biotin synthase from Arabidopsis thaliana. cDNA isolation and characterization of gene expression

The full-length BIO2 cDNA from Arabidopsis thaliana was isolated using an expressed sequence tag that was homologous to the Escherichia coli biotin synthase gene (BioB). Comparisons of the deduced amino acid sequence from BIO2 with bacterial and yeast biotin synthase homologs revealed a high degree of sequence similarity. The amino terminus of the predicted BIO2 protein contains a stretch of hydrophobic residues similar in composition to transit peptide sequences. BIO2 is a single-copy nuclear gene in Arabidopsis that is expressed at high levels in the tissues of immature plants. Expression of BIO2 was higher in the light relative to dark and was induced 5-fold during biotin-limited conditions. These results demonstrate that expression of at least one gene in this pathway is regulated in response to developmental, environmental, and bio-chemical stimuli.     

Receptor-like protein kinase genes of Arabidopsis thaliana

The isolation of a maize cDNA clone that encodes a membrane spanning protein kinase related to the self-incompatibility glycoproteins (SLG) of Brassica and structurally similar to the growth factor receptor tyrosine kinases has recently been reported. Three distinct receptor-like protein kinase (RLK) cDNA clones from Arabidopsis thaliana have now been identified. Two of the Arabidopsis RLK genes encode SLG-related protein kinases but have different patterns of expression: one is expressed predominantly in rosettes while the other is expressed primarily in roots. The third RLK gene contains an extracellular domain that consists of 21 leucine-rich repeats that are analogous to the leucine-rich repeats found in proteins from humans, flies and yeast. The Arabidopsis leucine-rich gene is expressed at equivalent levels in roots and rosettes. These results show that there are several genes in higher plants that encode members of the receptor protein kinase superfamily. The structural diversity and differential expression of these genes suggest that each plays a distinct and possibly important role in cellular signaling in plants.