Biochemical and biophysical properties of a highly active recombinant arginase from Leishmania (Leishmania) amazonensis and subcellular localization of native enzyme

Arginase (L-arginine amidinohydrolase, E.C. 3.5.3.1) is a metalloenzyme that catalyses the hydrolysis Of L-arginine to L-ornithine and urea. In Leishmania spp., the biological role of the enzyme may be involved in modulating NO production upon macrophage infection. Previously, we cloned and characterized the arginase gene from Leishmania (Leishmania) amazonensis. In the present work, we successfully expressed the recombinant enzyme in E. coli and performed biochemical and biophysical characterization of both the native and recombinant enzymes. We obtained K-M and V-max. values of 23.9(+/- 0.96) mM and 192.3 mu mol/min mg protein (+/- 14.3), respectively, for the native enzyme. For the recombinant counterpart, K-M was 21.5(+/- 0.90) mM and V-max was 144.9(+/- 8.9) mu mol/min mg. Antibody against the recombinant protein confirmed a glycosomal cellular localization of the enzyme in promastigotes. Data from light scattering and small angle X-ray scattering showed that a trimeric state is the active form of the protein. We determined empirically that a manganese wash at room temperature is the best condition to purify active enzyme. The interaction of the recombinant protein with the immobilized nickel also allowed us to confirm the structural disposition of histidine at positions 3 and 324. The determined structural parameters provide substantial data to facilitate the search for selective inhibitors of parasitic sources of arginase, which could subsequently point to a candidate for leishmaniasis therapy. (c) 2008 Elsevier B.V. All rights reserved.

AMP hydrolysis in soluble and microsomal rat cardiac cell fractions: kinetic characterization and molecular identification of 5 `-nucleotidase

The present study describes the enzymatic properties and molecular identification of 5`-nucleotidase in soluble and microsomal fractions from rat cardiac ventricles. Using AMP as a substrate, the results showed that the cation and the concentration required for maximal activity in the two fractions was magnesium at a final concentration of 1 mM. The pH optimum for both fractions was 9.5. The apparent K-m (Michaelis constant) values calculated from the Eadie-Hofstee plot were 59.7 +/- 10.4 mu M and 134.8 +/- 32.1 mu M, with V-max values of 6.7 +/- 0.4 and 143.8 +/- 23.8 nmol P-i/min/mg of protein (means +/- S.D., n = 4) from soluble and microsomal fractions respectively. Western blotting analysis of ecto-5`-nucleotidase revealed a 70 kDa protein in both fractions, with the major proportion present in the microsomal fraction. The presence of these enzymes in the heart probably has a physiological function in adenosine signalling. Furthermore, the presence of ecto-5`-nucleotidase in the microsomal fraction could have a role in the modulation of the excitation-contraction-coupling process through involvement of the Ca2+ influx into the sarcoplasmic reticulum. The measurement of maximal enzyme activities in the two fractions highlights the potential capacity of the different pathways of purine metabolism in the heart.

Biochemical characterization of serine acetyltransferase and cysteine desulfhydrase from Leishmania major

Cysteine metabolism exhibits atypical features in Leishmania parasites. The nucleotide sequence annotated as LmjF32.2640 encodes a cysteine desulfhydrase, which specifically catalyzes the breakdown of cysteine into pyruvate, NH(3) and H(2)S. Like in other pathogens, this capacity might be associated with regulatory mechanisms to control the intracellular level of cysteine, a highly toxic albeit essential amino acid, in addition to generate pyruvate for energy production. Besides, our results provide the first insight into the biochemical properties of Leishmania major serine acetyltransferase (SAT), which is likely involved in the two routes for de novo synthesis of cysteine in this pathogen. When compared with other members of SAT family, the N-terminal region of L. major homologue is uniquely extended, and seems to be essential for proper protein folding. Furthermore, unlike plant and bacterial enzymes, the carboxy-terminal-C(10) sequence stretch of L major SAT appears not to be implicated in forming a tight bi-enzyme complex with cysteine synthase. (C) 2010 Elsevier B.V. All rights reserved.

Characterization of proteinases from the midgut of Rhipicephalus (Boophilus) microplus involved in the generation of antimicrobial peptides

Background: Hemoglobin is a rich source of biologically active peptides, some of which are potent antimicrobials (hemocidins). A few hemocidins have been purified from the midgut contents of ticks. Nonetheless, how antimicrobials are generated in the tick midgut and their role in immunity is still poorly understood. Here we report, for the first time, the contribution of two midgut proteinases to the generation of hemocidins. Results: An aspartic proteinase, designated BmAP, was isolated from the midgut of Rhipicephalus (Boophilus) microplus using three chromatographic steps. Reverse transcription-quantitative polymerase chain reaction revealed that BmAP is restricted to the midgut. The other enzyme is a previously characterized midgut cathepsin L-like cysteine proteinase designated BmCL1. Substrate specificities of native BmAP and recombinant BmCL1 were mapped using a synthetic combinatorial peptide library and bovine hemoglobin. BmCL1 preferred substrates containing non-polar residues at P2 subsite and polar residues at P1, whereas BmAP hydrolysed substrates containing non-polar amino acids at P1 and P1`. Conclusions: BmAP and BmCL1 generate hemocidins from hemoglobin alpha and beta chains in vitro. We postulate that hemocidins may be important for the control of tick pathogens and midgut flora.

Cathepsin L-like genes of Trypanosoma vivax from Africa and South America - characterization, relationships and diagnostic implications

We characterized sequences from genes encoding cathepsin L-like (CatL-like) cysteine proteases from African and South American isolates of Trypanosoma vivax and T. vivax-like organisms, and evaluated their suitability as genetic markers for population structure analysis and diagnosis. Phylogenetic analysis of sequences corresponding to CatL-like catalytic domains revealed substantial polymorphism, and clades of sequences (TviCatL1-9) were separated by large genetic distances. TviCatL1-4 sequences were from cattle isolates from West Africa (Nigeria and Burkina Faso) and South America (Brazil and Venezuela), which belonged to the same T. vivax genotype. T. vivax-like genotypes from East Africa showed divergent sequences, including TviCatL5-7 for isolates from Mozambique and TviCatL8-9 for an isolate from Kenya. Phylogenetic analysis of CatL-like gene data supported the relationships among trypanosome species reflected in the phylogenies based on the analysis of small subunit (SSU) of ribosomal RNA gene sequence data. The discovery of different CatL-like sequences for each genotype, defined previously by ribosomal DNA data, indicate that these sequences provide useful targets for epidemiological and population genetic studies. Regions in CatL-like sequences shared by all T. vivax genotypes but not by other trypanosomes allowed the establishment of a specific and sensitive diagnostic PCR for epidemiological studies in South America and Africa. (C) 2008 Elsevier Ltd. All rights reserved.

Characterization of Paracoccidioides brasiliensis COX9, COX12, and COX16 respiratory genes

Paracoccidioides brasiliensis is a thermo-dimorphic fungus that is the causative agent of paracoccidioidomyicosis (PCM), a human systemic granulomatous mycosis found in Latin America. Dimorphic transition from mycelium to yeast is required for establishing pathogenicity. Dimorphism is marked by changes in mitochondrial physiology, including modulation of respiration rate. In this work, we present the identification of three P. brasiliensis nuclear genes PbCOX9, PbCOX12, and PbCOX16 that code for structural sub-units and a putative assembly facilitator (PbCOX16) of the mitochondrial cytochrome c oxidase (COX), the terminal enzyme complex of the respiratory chain. We measured their expression pattern during the dimorphic transition from mycelium to yeast and back by real-time reverse transcription quantitative polymerase chain reaction (real-time RT-qPCR). Our results show that messages from these genes increase during the mycelium to yeast transition and decrease during the opposite conversion. This result supports active mitochondrial participation in the transition. Heterologous complementation of the corresponding Saccharomyces cerevisiae null mutant with the PbCOX9 gene was successfully obtained. (C) 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Sugarcane Phosphoribosyl Pyrophosphate Synthetase: Molecular Characterization of a Phosphate-independent PRS

Phosphoribosyl pyrophosphate synthetase (PRS-EC:2.7.6.1) is an important enzyme present in several metabolic pathways, thus forming a complex family of isoenzymes. However, plant PRS enzymes have not been extensively investigated. In this study, a sugarcane prs gene has been characterized from the Sugar Cane Expressed Sequence Tag Genome Project. This gene contains a 984-bp open reading frame encoding a 328-amino acid protein. The predicted amino acid sequence has 77% and 78% amino acid sequence identity to Arabidopsis thaliana and Spinacia oleracea PRS4, respectively. The assignment of sugarcane PRS as a phosphate-independent PRS isoenzyme (Class II PRS) is verified following enzyme assay and phylogenetic reconstruction of PRS homologues. To gain further insight into the structural framework of the phosphate independence of sugarcane PRS, a molecular model is described. This model reveals the formation of two conserved domains elucidating the structural features involved in sugarcane PRS phosphate independence. The recombinant PRS retains secondary structure elements and a quaternary arrangement consistent with known PRS homologues, based on circular dichroism measurements.

Cloning, expression, purification and characterization of recombinant glutathione-S-transferase from Xylella fastidiosa

Xylella fastidiosa is an important pathogen bacterium transmitted by xylem-feedings leafhoppers that colonizes the xylem of plants and causes diseases on several important crops including citrus variegated chlorosis (CVC) in orange and lime trees. Glutathione-S-transferases (GST) form a group of multifunctional isoenzymes that catalyzes both glutathione (GSH)-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GSTs are the major detoxification enzymes found in the intracellular space and mainly in the cytosol from prokaryotes to mammals, and may be involved in the regulation of stress-activated signals by suppressing apoptosis signal-regulating kinase 1. In this study, we describe the cloning of the glutathione-S-transferase from X. fastidiosa into pET-28a(+) vector, its expression in Escherichia coli, purification and initial structural characterization. The purification of recombinant xfGST (rxfGST) to near homogeneity was achieved using affinity chromatography and size-exclusion chromatography (SEC). SEC demonstrated that rxfGST is a homodimer in solution. The secondary and tertiary structures of recombinant protein were analyzed by circular dichroism and fluorescence spectroscopy, respectively. The enzyme was assayed for activity and the results taken together indicated that rxfGST is a stable molecule, correctly folded, and highly active. Several members of the GST family have been extensively studied. However, xfGST is part of a less-studied subfamily which yet has not been structurally and biochemically characterized. In addition, these studies should provide a useful basis for future studies and biotechnological approaches of rxfGST. (C) 2008 Elsevier Inc. All rights reserved.

Purification, and Biochemical and Biophysical Characterization of Cellobiohydrolase I from Trichoderma harzianum IOC 3844

Because of its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum has a considerable potential in biomass hydrolysis applications. Trichoderma harzianum cellobiohydrolase I (ThCBHI), an exoglucanase, is an important enzyme in the process of cellulose degradation. Here, we report an easy single-step ion-exchange chromatographic method for purification of ThCBHI and its initial biophysical and biochemical characterization. The ThCBHI produced by induction with microcrystalline cellulose under submerged fermentation was purified on DEAE-Sephadex A-50 media and its identity was confirmed by mass spectrometry. The ThCBHI biochemical characterization showed that the protein has a molecular mass of 66 kDa and pi of 5.23. As confirmed by small-angle X-ray scattering (SAXS), both full-length ThCBHI and its catalytic core domain (CCD) obtained by digestion with papain are monomeric in solution. Secondary structure analysis of ThCBHI by circular dichroism revealed alpha-helices and beta-strands contents in the 28% and 38% range, respectively. The intrinsic fluorescence emission maximum of 337 nm was accounted for as different degrees of exposure of ThCBHI tryptophan residues to water. Moreover, ThCBHI displayed maximum activity at pH 5.0 and temperature of 50 degrees C with specific activities against Avicel and p-nitrophenyl-beta-D-cellobioside of 1.25 U/mg and 1.53 U/mg, respectively.

Molecular characterization of Mycobacterium massiliense and Mycobacterium bolletii in isolates collected from outbreaks of infections after laparoscopic surgeries and cosmetic procedures

An outbreak of infections affecting 311 patients who had undergone different invasive procedures occurred in 2004 and 2005 in the city of Belem, in the northern region of Brazil. Sixty-seven isolates were studied; 58 were from patients who had undergone laparoscopic surgeries, 1 was from a patient with a postinjection abscess, and 8 were from patients who had undergone mesotherapy. All isolates were rapidly growing nonpigmented mycobacteria and presented a pattern by PCR-restriction enzyme analysis of the hsp65 gene with BstEII of bands of 235 and 210 bp and with HaeIII of bands of 200, 70, 60, and 50 bp, which is common to Mycobacterium abscessus type 2, Mycobacterium bolletii, and Mycobacterium massiliense. hsp65 and. rpoB gene sequencing of a subset of 20 isolates was used to discriminate between these three species. hsp65 and rpoB sequences chosen at random from 11 of the 58 isolates from surgical patients and the postinjection abscess isolate presented the highest degrees of similarity with the corresponding sequences of M. massiliense. In the same way, the eight mesotherapy isolates were identified as M. bolletii. Molecular typing by pulsed-field gel electrophoresis (PFGE) grouped all 58 surgical isolates, while the mesotherapy isolates presented three different PFGE patterns and the postinjection abscess isolate showed a unique PFGE pattern. In conclusion, molecular techniques for identification and typing were essential for the discrimination of two concomitant outbreaks and one case, the postinjection abscess, not related to either outbreak all of which were originally attributed to a single strain of M. abscessus.

Digestive physiology and characterization of digestive cathepsin L-like proteinase from the sugarcane weevil Sphenophorus levis

Sugarcane is an important crop that has recently become subject to attacks from the weevil Sphenophorus levis, which is not efficiently controlled with chemical insecticides. This demands the development of new control devices for which digestive physiology data are needed. In the present study, ion-exchange chromatography of S. levis whole midgut homogenates, together with enzyme assays with natural and synthetic substrates and specific inhibitors, demonstrated that a cysteine proteinase is a major proteinase, trypsin is a minor one and chymotrypsin is probably negligible. Amylase, maltase and the cysteine proteinase occur in the gut contents and decrease throughout the midgut; trypsin is constant in the entire midgut, whereas a membrane-bound aminopeptidase predominates in the posterior midgut. The cysteine proteinase was purified to homogeneity through ion-exchange chromatography. The purified enzyme had a mass of 37 kDa and was able to hydrolyze Z-Phe-Arg-MCA and Z-Leu-Arg-MCA with k(cat)/K(m) values of 20.0 +/- 1.1 mu M(-1) s(-1) and 30.0 +/- 0.5 mu M(-1) s(-1), respectively, but not Z-Arg-Arg-MCA. The combined results suggest that protein digestion starts in the anterior midgut under the action of a cathepsin L-like proteinase and ends on the surface of posterior midgut cells. All starch digestion takes place in anterior midgut. These data will be instrumental to developing S. levis-resistant sugarcane. (C) 2011 Elsevier Ltd. All rights reserved.

Purification and partial characterization of an aminopeptidase from the midgut tissue of Dysdercus peruvianus

The surface of midgut cells in Hemiptera is ensheathed by a lipoprotein membrane (the perimicrovillar membrane), which delimits a closed compartment with the microvillar membrane, the so-called perimicrovillar space. In Dysdercus peruvianus midgut perimicrovillar space a soluble aminopeptidase maybe involved in the digestion of oligopeptides and proteins ingested in the diet. This D. peruvianus aminopeptidase was purified to homogeneity by ion-exchange chromatography on an Econo-Q column, hydrophobic interaction chromatography on phenyl-agarose column and preparative polyacrylamide gel electrophoresis. The results suggested that there is a single molecular species of aminopeptidase in D. peruvianus midgut. Molecular mass values for the aminopeptidase were estimated to be 106 kDa (gel filtration) and 55 kDa (SDS-PAGE), suggesting that the enzyme occurs as a dimer under native conditions. Kinetic data showed that D. peruvianus aminopeptidase hydrolyzes the synthetic substrates LpNA, RpNA, A beta NA and AsnMCA (K(m)s 0.65, 0.14, 0.68 and 0.74 mM, respectively). The aminopeptidase activity upon LpNA was inhibited by EDTA and 1,10-phenanthroline, indicating the importance of metal ions in enzyme catalysis. One partial sequence of BLAST-identified aminopeptidase was found by random sequencing of the D. peruvianus midgut cDNA library. Semi-quantitative RT-PCR analysis showed that the aminopeptidase genes were expressed throughout the midgut epithelium, in the epithelia of V1, V2 and V3. Malphigian tubules and fat body, but it was not expressed in the salivary glands. These results are important in furthering our understanding of the digestive process in this pest species. (c) 2010 Elsevier Inc. All rights reserved.

Characterization of a beta-1,3-glucanase active in the alkaline midgut of Spodoptera frugiperda larvae and its relation to beta-glucan-binding proteins

Spodoptera frugiperda beta-1,3-glucanase (SLam) was purified from larval midgut. It has a molecular mass of 37.5 kDa, an alkaline optimum pH of 9.0, is active against beta-1,3-glucan (laminarin), but cannot hydrolyze yeast beta-1,3-1,6-glucan or other polysaccharides. The enzyme is an endoglucanase with low processivity (0.4), and is not inhibited by high concentrations of substrate. In contrast to other digestive beta-1,3-glucanases from insects, SLam is unable to lyse Saccharomyces cerevisae cells. The cDNA encoding SLam was cloned and sequenced, showing that the protein belongs to glycosyl hydrolase family 16 as other insect glucanases and glucan-binding proteins. Multiple sequence alignment of beta-1,3-glucanases and beta-glucan-binding protein supports the assumption that the beta-1,3-glucanase gene duplicated in the ancestor of mollusks and arthropods. One copy originated the derived beta-1,3-glucanases by the loss of an extended N-terminal region and the beta-glucan-binding proteins by the loss of the catalytic residues. SLam homology modeling suggests that E228 may affect the ionization of the catalytic residues, thus displacing the enzyme pH optimum. SLam antiserum reacts with a single protein in the insect midgut. Immunocytolocalization shows that the enzyme is present in secretory vesicles and glycocalyx from columnar cells. (C) 2010 Elsevier Ltd. All rights reserved.

Molecular characterization of nitrate reductase gene and its expression in the marine red alga Gracilaria tenuistipitata (Rhodophyta)

The enzyme nitrate reductase (NR) responsible for the conversion of nitrate to nitrite is considered to be the rate-limiting step in nitrogen assimilation. The economically important marine macroalga Gracilaria tenuistipitata presents a circadian oscillation in NR protein content and activity. In order to identify if the regulation of NR in G. tenuistipitata happens at transcriptional levels, the NR cDNA and gene were sequenced and the NR mRNA expression was studied. Analysis of the sequenced gene revealed absence of introns which is unusual for NR genes. The transcriptional profiling revealed a circadian rhythm for NR; furthermore, a rhythm was observed in constant light condition, suggesting a possible regulation by the biological clock at the mRNA levels for NR in G. tenuistipitata.

Purification and partial characterization of a midgut membrane-bound alpha-glucosidase from Quesada gigas (Hemiptera: Cicadidae)

Adults of Quesada gigas (Hemiptera: Cicadidae) have a major alpha-glucosidase bound to the perimicrovillar membranes, which are lipoprotein membranes that surround the midgut cell microvilli in Hemiptera and Thysanoptera. Determination of the spatial distribution of alpha-glucosidases in Q. gigas midgut showed that this activity is not equally distributed between soluble and membrane-bound isoforms. The major membrane-bound enzyme was solubilized in the detergent Triton X-100 and purified to homogeneity by means of gel filtration on Sephacryl S-100, and ion-exchange on High Q and Mono Q columns. The purified alpha-glucosidase is a protein with a pH optimum of 6.0 against the synthetic substrate p-nitrophenyl alpha-D-glucoside and M(r) of 61,000 (SDS-PAGE). Taking into account V(Max)/K(M) ratios, the enzyme is more active on maltose than sucrose and prefers oligomaltodextrins up to maltopentaose, with lower efficiency for longer chain maltodextrins. The Q gigas alpha-glucosidase was immunolocalized in perimicrovillar membranes by using a monospecific polyclonal antibody raised against the purified enzyme from Dysdercus peruvianus. The role of this enzyme in xylem fluid digestion and its possible involvement in osmoregulation is discussed. (C) 2009 Elsevier Inc. All rights reserved.