Study of peripheral domains in structure-function of isocitrate lyase (ICL) from Pseudomonas aeruginosa.

The capacity of Pseudomonas aeruginosa to assimilate nutrients is essential for niche colonization and contributes to its pathogenicity. Isocitrate lyase (ICL), the first enzyme of the glyoxylate cycle, redirects isocitrate from the tricarboxylic acid cycle to render glyoxylate and succinate. P. aeruginosa ICL (PaICL) is regarded as a virulence factor due to its role in carbon assimilation during infection. The AceA/ICL protein family shares the catalytic domain I, triosephosphate isomerase barrel (TIM-barrel). The carboxyl terminus of domain I is essential for Escherichia coli ICL (EcICL) of subfamily 1. PaICL, which belongs to subfamily 3, has domain II inserted at the periphery of domain I, which is believed to participate in enzyme oligomerization. In addition, PaICL has the α13-loop-α14 (extended motif), which protrudes from the enzyme core, being of unknown function. This study investigates the role of domain II, the extended motif, and the carboxyl-terminus (C-ICL) and amino-terminus (N-ICL) regions in the function of the PaICL enzyme, also as their involvement in the virulence of P. aeruginosa PAO1. Deletion of domain II and the extended motif results in enzyme inactivation and structural instability of the enzyme. The His6-tag fusion at the C-ICL protein produced a less efficient enzyme than fusion at the N-ICL, but without affecting the acetate assimilation or virulence. The PaICL homotetrameric structure of the enzyme was more stable in the N-His6-ICL than in the C-His6-ICL, suggesting that the C-terminus is critical for the ICL quaternary conformation. The ICL-mutant A39 complemented with the recombinant proteins N-His6-ICL or C-His6-ICL were more virulent than the WT PAO1 strain. The findings indicate that the domain II and the extended motif are essential for the ICL structure/function, and the C-terminus is involved in its quaternary structure conformation, confirming that in P. aeruginosa, the ICL is essential for acetate assimilation and virulence.

Physiological and enzymatic alterations in sesame seeds submitted to different osmotic potentials.

With the imminence of global climate changes that affect the temperature and the rainfall uniformity, it is growing the concern about the adaptation of crops to the water deficit. Thus, the objective of this study was to evaluate alterations in physiological and enzymatic mechanisms during the germination process of sesame seeds under different water availability. To simulate the water restriction we used PEG6000, a high molecular weight molecule that does not penetrate the seed structure but allows different osmotic potentials. The treatments were -0.1, -0.2, and -0.3 MPa, and the control. Germination, first-count germination, germination velocity index, and length and dry mass of the hypocotyl and radicle were performed. The seeds were weighed before and after treatments every 3 h. After each weighing, 100 seeds were taken for analysis of the enzymes alcohol dehydrogenase (ADH), malate dehydrogenase, esterase, catalase (CAT), superoxide dismutase (SOD), isocitrate lyase (ICL), and glutamate dehydrogenase (GTDH). The statistical design was completely randomized with five replications. PEG6000 prolonged ADH activity during the beginning of germination, maintaining the anaerobic metabolism for longer. Subsequently, their activity was reduced, as well as ICL, favoring the deterioration of the seeds that take the time to germinate. Behavior was evidenced by the appearance of SOD, CAT, and GTDH isoforms after 24 h of imbibition when water restriction was imposed. Therefore, the PEG600 is efficient in simulating water deficit conditions in future scenarios of climate change, offering impotent information regarding the germination behavior of the plants under these conditions.

Physiological quality and gene expression during the development of habanero pepper (Capsicum chinense Jacquin) seeds.

Phytohormones have different characteristics and functions, and they may be subject to changes in their gene expression and synthesis during seed development. In this study, we evaluated the physiological qualities of habanero peppers (Capsicum chinense Jacquin) during seed development and the expression of genes involved in germination. Seeds were obtained from fruits harvested at different stages of development [i.e., 14, 21, 28, 35, 42, 49, 56, 63, and 70 days after anthesis (DAA)]. Immediately after harvesting, the seeds were subjected to various tests to determine moisture content, germination, first count germination, and seedling emergence. Real-time polymerase chain reaction was used to evaluate the expression of various genes, including MAN2, NCED, B73, ICL6, and GA3ox. Electrophoresis was used to assess the expression of various enzymes, including α-amylase, isocitrate-lyase, and endo-ß-mannanase. Habanero peppers harvested at 70 DAA and subjected to 7 days of rest exhibited higher germination rates and vigor compared to those harvested at all other developmental stages. Peppers harvested at 63 DAA without drying exhibited higher α amylase and AmyB73 gene expression levels. Peppers harvested at 70 DAA with 7 days of rest exhibited higher endo-ß-mannanase expression levels. MAN2 gene expression increased during the development of non-dried seeds until 70 DAA. Peppers harvested at 42 DAA exhibited the highest isocitrate-lyase and ICL6 gene activity levels in comparison to those at all other developmental stages.

Inhibition of Paracoccidioides lutzii Pb01 isocitrate lyase by the natural compound argentilactone and its semi-synthetic derivatives.

The dimorphic fungus Paracoccidioides spp. is responsible for paracoccidioidomycosis, the most prevalent systemic mycosis in Latin America, causing serious public health problems. Adequate treatment of mycotic infections is difficult, since fungi are eukaryotic organisms with a structure and metabolism similar to those of eukaryotic hosts. In this way, specific fungus targets have become important to search of new antifungal compound. The role of the glyoxylate cycle and its enzymes in microbial virulence has been reported in many fungal pathogens, including Paracoccidioides spp. Here, we show the action of argentilactone and its semi-synthetic derivative reduced argentilactone on recombinant and native isocitrate lyase from Paracoccidioides lutzii Pb01 (PbICL) in the presence of different carbon sources, acetate and glucose. Additionally, argentilactone and its semi-synthetic derivative reduced argentilactone exhibited relevant inhibitory activity against P. lutzii Pb01 yeast cells and dose-dependently influenced the transition from the mycelium to yeast phase. The other oxygenated derivatives tested, epoxy argentilactone and diol argentilactone-, did not show inhibitory action on the fungus. The results were supported by in silico experiments.

Residues Asn214, Gln211, Glu219 and Gln221 contained in the subfamily 3 catalytic signature of the isocitrate lyase from Pseudomonas aeruginosa are involved in its catalytic and thermal properties.

Isocitrate lyase, encoded by the aceA gene, plays an important role in the ability of Pseudomonas aeruginosa to grow on fatty acids, acetate, acyclic terpenes, and amino acids. Phylogenetic analysis indicated that the ICL superfamily is divided in two families: the ICL family, which includes five subfamilies, and the 2-methylisocitrate lyase (MICL) family. ICL from P. aeruginosa (ICL-Pa) was identified in a different ICL node (subfamily 3) than other Pseudomonas ICL enzymes (grouped in subfamily 1). Analysis also showed that psychrophilic bacteria are mainly grouped in ICL subfamily 3, whose ICL proteins contain the highly conserved catalytic pattern QIENQVSDEKQCGHQD. We performed site-directed mutagenesis, enzymatic activity, and structure modeling of conserved residues in mutated ICLs by using ICL-Pa as a model. Our results indicated that the N214 residue is essential for catalytic function, while mutating the Q211, E219, and Q221 residues impairs its catalytic and thermostability properties. Our findings suggest that conserved residues in the subfamily 3 signature of ICL-Pa play important roles in catalysis and thermostability and are likely associated with the catalytic loop structural conformation.

Bahamaolides A and B, antifungal polyene polyol macrolides from the marine actinomycete Streptomyces sp.

Bahamaolides A and B (1 and 2), two new 36-membered macrocyclic lactones, were isolated from the culture of the marine actinomycete Streptomyces sp. derived from a sediment sample collected at North Cat Cay in the Bahamas. The planar structures of 1 and 2, bearing a hexaenone and nine consecutive skipped hydroxy groups, were determined by 1D and 2D NMR, mass, IR, and UV spectra. The absolute configurations of the bahamaolides were established by combined multistep chemical reactions and spectroscopic analysis. Bahamaolide A displayed significant inhibitory activity against Candida albicans isocitrate lyase and antifungal activity against various pathogenic fungi.

Phosphorylation is the major mechanism regulating isocitrate lyase activity in Paracoccidioides brasiliensis yeast cells.

The glyoxylate cycle plays an essential role for anaplerosis of oxaloacetate during growth of microorganisms on carbon sources such as acetate or fatty acids and has been shown to contribute to virulence of several pathogens. Here, we investigated the transcriptional and post-translational regulation of the glyoxylate cycle key enzyme isocitrate lyase (PbICL) in the human pathogenic fungus Paracoccidioides brasiliensis. Although sequence analyses on fungal isocitrate lyases revealed a high phylogenetic conservation, their regulation seems to differ significantly. Closely related Aspergillus species regulate the glyoxylate cycle at the transcriptional level, whereas Pbicl was constitutively expressed in yeast cells. However, only low PbICL activity was detected when cells were grown in the presence of glucose. Two-dimensional gel analyses with subsequent antibody hybridization revealed constitutive production of PbICL, but low PbICL activity on glucose coincided with extensive protein phosphorylation. Since an in vitro dephosphorylation of PbICL from glucose grown cells strongly increased ICL activity and resembled the phosphorylation pattern of highly active acetate grown cells, post-translational modification seems the main mechanism regulating PbICL activity in yeast cells. In agreement, a transfer of yeast cells from glucose to acetate medium increased PbICL activity without requirement of de novo protein synthesis. Thus, inactivation of PbICL by phosphorylation is reversible, denoting a new strategy for the rapid adaptation to changing environmental conditions.

Identification of the aceA gene encoding isocitrate lyase required for the growth of Pseudomonas aeruginosa on acetate, acyclic terpenes and leucine.

Pseudomonas aeruginosa PAO1 mutants affected in acyclic monoterpenes, n-octanol, and acetate assimilation were isolated using transposon mutagenesis. The isocitrate lyase gene (aceA) corresponding to ORF PA2634 of the PAO1 strain genome was identified in one of these mutants. The aceA gene encodes a protein that is 72% identical to the isocitrate lyase (ICL) characterized from Colwellia maris, but is less than 30% identical to their homologues from pseudomonads. The genetic arrangement of aceA suggests that it is a monocistronic gene, and no adjacent related genes were found. The ICL protein was detected as a 60-kDa band in sodium dodecyl sulfate polyacrylamide gel electrophoresis from cultures grown on acetate, but not in glucose-grown PAO1 cultures. Genetic complementation further confirmed that the aceA gene encodes the ICL enzyme. The ICL enzyme activity in crude extracts from cultures of the PAO1 strain was induced by acetate, citronellol and leucine, and repressed by growth on glucose or citrate. These results suggest that ICL is involved in the assimilation of acetate, acyclic monoterpenes of the citronellol family, alkanols, and leucine, in which the final intermediary acetyl-coenzyme A may be channelled to the glyoxylate shunt.

The ICL1 gene of Pichia pastoris, transcriptional regulation and use of its promoter.

We cloned and characterized a gene encoding isocitrate lyase from the methylotrophic yeast Pichia pastoris. This gene was isolated from a P. pastoris genomic library using a homologous PCR hybridization probe, amplified with two sets of degenerate primers designed from conserved regions in yeast isocitrate lyases. The cloned gene was sequenced and consists of an open reading frame of 1563 bp encoding a protein of 551 amino acids. The molecular mass of the protein is calculated to be 60.6 kDa with high sequence similarity to isocitrate lyase from other organisms. There is a 64% identity between amino acid sequences of P. pastoris Icl and Saccharomyces cerevisiae Icl. Northern blot analyses showed that, as in S. cerevisiae, the steady-state ICL1 mRNA levels depend on the carbon source used for cell growth. Expression in P. pastoris of the dextranase gene (dexA) from Penicillium minioluteum under control of the ICL1 promoter proved that P(ICL1) is a good alternative for the expression of heterologous proteins in this methylotrophic yeast. The sequence presented here has been deposited in the EMBL data library under Accession No. AJ272040.

[Effect of trace metals on cell morphology, enzyme activation, and production of citric acid in a strain of Aspergillus wentii]. / Efecto de metales traza sobre la morfología celular, actividad enzimática y producción de ácido cítrico en una cepa de Aspergillus wentii.

Data concerning the effect of very low concentrations of metals on citric acid production by microorganisms, as well as on the activity of enzymes presumptively involved in the process, are confuse. The bulk of information was obtained mainly studying selected strains of Aspergillus niger. Information concerning other citric acid producer filamentous fungi, such as A. wentii, is scanty. In the present article we report the effect of different cations on the growth pattern of A. wentii P1 as well as on the related citric acid production and the activity of several enzymes. It was found that without any addition to the culture medium the fungus developed a pelleted form of growth, pellets being about 1.5 mm in diameter. The citric acid yield was about 90%. The addition of Cu2+ impaired the sugar uptake, as well as the production of citric acid and biomass. The uptake of sugar increased in the presence of Zn2+, and there was a marked increase of the biomass production, which could account for the low citric acid production. The addition of Fe2+ impaired the citric acid production and, as sulfate, the sugar uptake. The presence of Fe3+ markedly impaired the citric acid production and increased the sugar uptake. There is no agreement about the enzymes involved in the accumulation of citric acid by microorganisms. In spite of this, aconitase (Ac), isocitrate lyase (IL), isocitrate dehydrogenase NAD(+)-dependent (ICDH- NAD+) and isocitrate dehydrogenase NADP(+)-dependent (ICDH-NADP+) are often postulated as key enzymes. In our case, these enzymes were active during the standard fermentation, although with variations, particularly concerning Ac and IL. The behavior of enzymes might be different when tested in vivo or in vitro, mainly from the quantitative point of view. Nevertheless, the activity determined in vitro might give some indication concerning the effect on fermentation of substances present in the medium. It was found that all the enzymes tested increased their activity in the presence of Fe2+ and Fe3+. The addition of Cu2+ improved the activity of Ac, IL and ICDH-NADP+, while that of ICDH-NAD+ was impaired. The presence of Zn2+ decreased the activity of the enzymes excepting that of ICDH-NADP+ which increased. It must be pointed out that according to these results there are differences concerning the behavior of ICDH-NAD+ and ICDH-NADP+ in the presence of Cu2+ or Zn2+.

Efecto de metales traza sobre la morfología celular, actividad enzimßtica y producción de ácido cítrico en una cepa de Aspergillus wentii / Effect of trace metals on cell morphology, enzyme activation, and production of citric acid in a strain of Aspergillus wentii

Data concerning the effect of very low concentrations of metals on citric acid production by microorganisms, as well as on the activity of enzymes presumptively involved in the process, are confuse. The bulk of information was obtained mainly studying selected strains of Aspergillus niger. Information concerning other citric acid producer filamentous fungi, such as A. wentii, is scanty. In the present article we report the effect of different cations on the growth pattern of A. wentii P1 as well as on the related citric acid production and the activity of several enzymes. It was found that without any addition to the culture medium the fungus developed a pelleted form of growth, pellets being about 1.5 mm in diameter. The citric acid yield was about 90. The addition of Cu2+ impaired the sugar uptake, as well as the production of citric acid and biomass. The uptake of sugar increased in the presence of Zn2+, and there was a marked increase of the biomass production, which could account for the low citric acid production. The addition of Fe2+ impaired the citric acid production and, as sulfate, the sugar uptake. The presence of Fe3+ markedly impaired the citric acid production and increased the sugar uptake. There is no agreement about the enzymes involved in the accumulation of citric acid by microorganisms. In spite of this, aconitase (Ac), isocitrate lyase (IL), isocitrate dehydrogenase NAD(+)-dependent (ICDH- NAD+) and isocitrate dehydrogenase NADP(+)-dependent (ICDH-NADP+) are often postulated as key enzymes. In our case, these enzymes were active during the standard fermentation, although with variations, particularly concerning Ac and IL. The behavior of enzymes might be different when tested in vivo or in vitro, mainly from the quantitative point of view. Nevertheless, the activity determined in vitro might give some indication concerning the effect on fermentation of substances present in the medium. It was found that all the enzymes tested increased their activity in the presence of Fe2+ and Fe3+. The addition of Cu2+ improved the activity of Ac, IL and ICDH-NADP+, while that of ICDH-NAD+ was impaired. The presence of Zn2+ decreased the activity of the enzymes excepting that of ICDH-NADP+ which increased. It must be pointed out that according to these results there are differences concerning the behavior of ICDH-NAD+ and ICDH-NADP+ in the presence of Cu2+ or Zn2+.(AU)

Effect of mercuric chloride on the activities of some hepatic enzymes of regenerating rabbit liver following partial hepatectomy.

Mercuric chloride (HgCl2) administered at a dose of 1mg/kg body wt/day for 5 days decreased hepatic lactate dehydrogenase (LDH) activity (63%) and increased isocitrate dehydrogenase (ICDH) activity (127%). After withdrawal of HgCl2 treatment for 10 days, the LDH and glutamate pyruvate transaminase (GPT) activities showed 56% and 40% decrease, respectively, while alkaline phosphatase (AkP) activity increased 4.12 fold. The ICDH activity got normalized. Glutamate oxaloacetate transaminase (GDT) was not affected at all. The hepatic LDH, ICDH and GPT activities decreased 58%, 72% and 82%, respectively, five days after partial hepatectomy (PH), while AkP activity increased 90%. At the end of 15 days after PH, the hepatic ICDH activity increased 4.27 fold, while GPT and GOT activities decreased 67% and 91%, respectively. The hepatic ICDH activity of PH-rabbit increased 53%, after 5 days of HgCl2 treatment post-PH, while GPT and AkP activities decreased 89% and 97%, respectively, during this period. The ICDH activity increased 416%, 10 days after the last dose, while all other enzymes showed normal values. The total body growth rate and relative liver weight decreased under all experimental conditions.

Atividade da isocitrato liase em Mucor rouxii / Activity of isocitrate liase in mucor rouxii

O ficomiceto dimórfico Mucor rouxii foi cultivado em meio que continha glucose a 2% (YPG), tendo-se verificado um crescimento bifásico característico. A isocitrato liase, enzima do ciclo do glioxilato, apareceu sob estas condiçöes somente após a glucose do meio ter sido totalmente esgotada. Altos níveis de atividade da isocitrato liase foram encontrados durante o metabolismo gluconeogenético. Isso mostra a participaçäo do ciclo do glioxilato como fonte de succinato e de oxalacetato para as vias biossintéticas do fungo. Em meio complexo sem glucose (YP), estritamente gluconeogenético, foram encontradas altas atividades de isocitrato liase