Plant Peroxisomes: Molecular Basis of the Regulation of their Functions

β -oxidation, the glyoxylate cycle and the glycolate pathway for photorespiration. Recent molecular biological studies have revealed that most of these enzymes possess either one of two peroxisomal targeting signals (PTS) within their amino acid sequence. One of the signals, PTS1, is found at the carboxy-terminus, while the other, PTS2, is found within the amino-terminal presequence. Subsequent to the synthesis and folding of these enzymes in the cytosol, the targeting signal in the folded proteins may bind to the corresponding receptors. At present, only a receptor that recognizes PTS1 has been identified in higher plants. After the binding of the protein and the receptor, the protein complex may be recognized by docking proteins that exist in the peroxisomal membrane. The mechanisms responsible for the recognition of peroxisomal proteins are now under investigation. Genetic analyses of Arabidopsis mutants with defective peroxisomes may give us some clues to understanding the mechanisms of peroxisomal protein import. Received 18 November 1999/ Accepted in revised form 13 January 2000     

Inhibition of purine utilization by adenine in Alcaligenes eutrophus H16

With 0.5% substrate present in mineral medium, cells of Alcaligenes eutrophus H 16 were able to grow heterotrophically at the expense of guanine, hypoxanthine and xanthine, but not of adenine as sole sources of carbon and nitrogen. An increase in cell counts, however, was observed at lower adenine concentrations (0.1%). Similarly, adenine was only respired if present at low concentrations. Higher amounts of adenine were inhibitory to the utilization of adenine, guanine, hypoxanthine, xanthine, allantoin and glyoxylate, but not to that of fructose or glycerate. The adenine-dependent inhibition of adenine utilization was not overcome by the addition of thiamine, uridine or cytidine. The enzyme glyoxylate carboligase, usually formed in presence of metabolisable purines and of allantoin, was synthesized only at low adenine concentrations. Higher amounts were inhibitory even with allantoin present as additional substrate. According to these resutls, the utilization of purine derivatives and of allantoin as sources of carbon and energy is repressed by adenine in cells of A. eutrophus H 16.     

Bacteria and archaea as the sources of traits for enhanced plant phenotypes

Rising global demand for food and population increases are driving the need for improved crop productivity over the next 30 years. Plants have inherent metabolic limitations on productivity such as inefficiencies in carbon fixation and sensitivity to environmental conditions. Bacteria and archaea inhabit some of the most inhospitable environments on the planet and possess unique metabolic pathways and genes to cope with these conditions. Microbial genes involved in carbon fixation, abiotic stress tolerance, and nutrient acquisition have been utilized in plants to enhance plant phenotypes by increasing yield, photosynthesis, and abiotic stress tolerance. Transgenic plants expressing bacterial and archaeal genes will be discussed along with emerging strategies and tools to increase plant growth and yield.     

Gel mobility shift scanning of the acetate-inducible promoters fromNeurospora crassa reveals a common co-inducible DNA-binding protein

The promoter regions of four acetate-inducible genes ofNeurospora crassa, acu-3, acu-5, acu-8 andacu-9, have been sequenced. Using a scanning gel mobility shift assay particular DNA regions in each promoter have been shown specifically to bind partially purified protein extracted from acetate-induced mycelia. The protein-binding regions so defined have common sequence motifs, elements of which are similar to those required for acetate induction inAspergillus nidulans.     

Control of photosynthesis in barley mutants with reduced activities of glutamine synthetase and glutamate synthase

Heterozygous mutants of barley (Hordeum vulgare L. cv. Maris Mink) with decreased activities of chloroplastic glutamine synthetase (GS) between 97 and 47% of the wild type and ferredoxin dependent glutamate synthase (Fd-GOGAT) down to 64% of the wild type have been used to study aspects of glyoxylate metabolism and the effect of glyoxylate on the activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in vivo. In the leaf, the extractable activities of serine:glyoxylate aminotransferase decreased with a decrease in GS whereas activities of glutamate and alanine:glyoxylate aminotransferase increased, pointing to a re direction of amino donors from serine to glutamate and alanine. Under ambient conditions, the leaf contents of glutamate and alanine declined continuously with a decrease in GS, in parallel with the decrease in total amino acids. Glycine, serine and asparagine contents decreased with a decrease in GS to approximately 70% of the wild type, but increased again with a further decrease in GS. At high irradiances and at low CO₂ concentrations, glyoxylate contents exhibited a pronounced minimum between 60% and 80% GS. With a further decrease in GS, glyoxylate contents recovered and approached values similar to the wild type. The activation state of Rubisco showed a negative correlation with glyoxylate contents, indicating that a decrease in GS feeds back on the first step of carbon assimilation and photorespiration. The activation state of stromal fructose-1,6-bisphosphatase was unaffected by a decrease in GS or Fd-GOGAT, whereas the activation state of NADP dependent malate dehydrogenase changed in a complex manner. The CO₂photocompensation point, ^*, was appreciably increased in mutants with 47% GS. Mitochondrial respiration in the light (R_d) was reduced with a decrease in GS. Relative rates of CO₂ release into CO₂-free air between the wild type and the 47%-GS mutant correlated with determinations of ^*. These data are consistent with the view that when GS is decreased there is an increased oxidative decarboxylation of glyoxylate resulting from a decreased availability of amino donors for the transamination of glyoxylate to glycine, and that when GS activities are lower than 70% of the wild type an additional mechanism operates to reduce the photorespiratory loss of ammonia.Abbreviations AGAT nine:glyoxylate aminotransferase - FBPase fructose-1,6-bisphosphatase - Fd-GOGAT ferredoxin dependent glutamate synthase - GGAT glutamate:glyoxylate aminotransferase - GS glutamine synthetase - MDH malate dehydrogenase - PFD photon flux density - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - SGAT serine:glyoxylate aminotransferase This research was supported by the Biotechnology and Biological Sciences Research Council initiative on the Biochemistry of Metabolic Regulation in Plants (PG 50/555).     

Metabolic flux analyses of Pseudomonas aeruginosa cystic fibrosis isolates

Pseudomonas aeruginosa is a metabolically versatile wide-ranging opportunistic pathogen. In humans P. aeruginosa causes infections of the skin, urinary tract, blood, and the lungs of Cystic Fibrosis patients. In addition, P. aeruginosa's broad environmental distribution, relatedness to biotechnologically useful species, and ability to form biofilms have made it the focus of considerable interest. We used ¹³C metabolic flux analysis (MFA) and flux balance analysis to understand energy and redox production and consumption and to explore the metabolic phenotypes of one reference strain and five strains isolated from the lungs of cystic fibrosis patients. Our results highlight the importance of the oxidative pentose phosphate and Entner-Doudoroff pathways in P. aeruginosa growth. Among clinical strains we report two divergent metabolic strategies and identify changes between genetically related strains that have emerged during a chronic infection of the same patient. MFA revealed that the magnitude of fluxes through the glyoxylate cycle correlates with growth rates.     

Metabolism and decarboxylation of glycollate and serine in leaf peroxisomes

The linked utilization of glycollate and L-serine has been studied in peroxisomal preparations from leaves of spinach beet (Beta vulgaris L.). The generation of glycine from glycollate was found to be balanced by the production of hydroxypyruvate from serine and similarly by 2-oxoglutarate when L-glutamate was substituted for L-serine. In the presence of L-malate and catalytic quantities of NAD⁺, about 40% of the hydroxypyruvate was converted further to glycerate, whereas with substrate quantities of NADH, this conversion was almost quantitative. CO₂ was released from the carboxyl groups of both glycollate and serine. Since the decarboxylation of both substrates was greatly in creased by the catalase inhibitor, 3-amino-1,2,4-triazole, and abolished by bovine liver catalase, it was attributed to the nonenzymic attack of H₂O₂, generated in glycollate oxidation, upon glyoxylate and hydroxypyruvate respectively. At 25–30° C, about 10% of the glyoxylate and hydroxypyruvate accumulated was decarboxylated, and the release of CO₂ from each keto-acid was related to the amounts present. It is suggested that hydroxypyruvate decarboxylation might contribute significantly to photorespiration and provide a metabolic route for the complete oxidation of glycollate, the magnitude of this contribution depending upon the concentrations of glyoxylate and hydroxypyruvate in the peroxisomes.     

Characterization of a malate dehydrogenase in the cyanobacterium Coccochloris peniocystis

A malate dehydrogenase (MDH) was characterized from the cyanobacterium Coccochloris peniocystis. The enzyme was purified approximately 180-fold and had a molecular weight of about 90000. The enzyme had a pH optimum of pH 6.7 to 7.5; a K_m (malate) of 5.6 mM and K_ms for NAD and NADP of 24 M and 178 M, respectively, although similar V_max were obtained with either pyridine nucleotide. Enzyme activity was inhibited by ATP, citrate, oxalacetate, acetyl CoA and CoA. Enzyme assays with uniformly ¹⁴C-labelled malate caused no ¹⁴CO₂ release, indicating this MDH is not a malic enzyme. Electrophoresis and S-200 gel filtration of the partially purified enzyme indicated a single MDH was present in this preparation. A second, less abundant, MDH was present in crude extracts. The presence of MDH in this organism is consistent with the operation of a glyoxylate cycle which, in the absence of a TCA cycle, would provide organic acids required in secondary carbon metabolism. ATP inhibition of MDH may allow for light regulation of MDH activity since, in the light, oxaloacetic acid is generated by phosphoenolpyruvate carboxylase activity.Abbreviations MDH malate dehydrogenase - PEPcase phosphoenolpyruvate carboxylase - MOPS 3-[N-Morpholino] propane sulfonic acid - TRIS Tris(hydroxymethyl)-aminomethane - EDTA Disodium Ethylenadiamine Tetraacetate - MES 2[N-Morpholino]-ethane Sulfonic Acid - EPPS N-2-Hydroxyethylpiperazine Propane - MW Molecular weight - OAA Oxaloacetic acid     

A comparison of the phosphorylated and unphosphorylated forms of isocitrate dehydrogenase from Escherichia coli ML308

NADP+ can protect active isocitrate dehydrogenase against attack by several proteases. Inactive phosphorylated isocitrate dehydrogenase is much less susceptible to proteolysis than the active enzyme, and it is not protected by NADP+. The results suggest that binding of NADP+ to, or phosphorylation of, active isocitrate dehydrogenase induces similar conformational states. Fluorescence titration experiments show that NADPH can bind to active but not to inactive isocitrate dehydrogenase. It is suggested that the phosphorylation of isocitrate dehydrogenase may occur close to its coenzyme binding site.     

Enzyme profiles of Thiobacillus versutus after aerobic and denitrifying growth: Regulation of isocitrate lyase

The specific activities of the tricarboxylic acid (TCA) cycle enzymes in Thiobacillus versutus were invariably lower after aerobic growth as compared to denitrifying growth in acetate- or succinate-limited chemostat cultures. Of the glyoxylate cycle enzymes, isocitrate lyase (ICL) activity was nil during aerobic and 76 nmol·min^-1·mg^-1 protein during denitrifying growth on acetate whereas malate synthase (MS) did not change. In succinate-grown cells ICL was always near nil. The change in ICL and MS was followed after pulse additions of acetate and nitrate to an aerobic acetate-limited chemostat culture made anaerobic prior to the first pulse. ICL remained nil during denitrifying growth after the first pulse but increased to 47 and 81 nmol ·min^-1·mg^-1 protein after the second and third pulse, respectively. MS remained unaltered. The appearance of ICL was dependent upon de novo protein synthesis. During transition in a steady state culture on acetate from oxygen to nitrate as terminal electron acceptor, denitrifying growth started after 0.6 volume replacements. The resumption of growth was concomitant with an increase in TCA cycle enzyme activities. ICL was observed only after two volume replacements. During the reverse transition, ICL disappeared at a rate twice the dilution rate. SDS polyacrylamide gelectrophoresis of cell-free extracts containing ICL showed a major protein band with a R_f value identical to purified ICL and a mol·wt. of 60,000. ICL from T. versutus was inhibited by 1.5 mM itaconate but not by 10 mM phosphoenolpyruvate. Its activity was dependent upon the presence of Mg²⁺ and cysteine.Abbreviations TCA Tricarboxylic acid - ICL isocitrate lyase - MS malate synthase - FPLC fast protein liquid chromatography - maximum specific oxygen consumption rate