Crystal structure of Leishmania tarentolae hypoxanthine-guanine phosphoribosyltransferase

Abstract Background Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 2.4.2.8) is a central enzyme in the purine recycling pathway. Parasitic protozoa of the order Kinetoplastida cannot synthesize purines de novo and use the salvage pathway to synthesize purine bases, making this an attractive target for antiparasitic drug design. Results The glycosomal HGPRT from Leishmania tarentolae in a catalytically active form purified and co-crystallized with a guanosine monophosphate (GMP) in the active site. The dimeric structure of HGPRT has been solved by molecular replacement and refined against data extending to 2.1 Å resolution. The structure reveals the contacts of the active site residues with GMP. Conclusion Comparative analysis of the active sites of Leishmania and human HGPRT revealed subtle differences in the position of the ligand and its interaction with the active site residues, which could be responsible for the different reactivities of the enzymes to allopurinol reported in the literature. The solution and analysis of the structure of Leishmania HGPRT may contribute to further investigations leading to a full understanding of this important enzyme family in protozoan parasites.

Unraveling the structure of sugarcane bagasse after soaking in concentrated aqueous ammonia (SCAA) and ethanol production by Scheffersomyces (Pichia) stipitis

Abstract Background Fuel ethanol production from sustainable and largely abundant agro-residues such as sugarcane bagasse (SB) provides long term, geopolitical and strategic benefits. Pretreatment of SB is an inevitable process for improved saccharification of cell wall carbohydrates. Recently, ammonium hydroxide-based pretreatment technologies have gained significance as an effective and economical pretreatment strategy. We hypothesized that soaking in concentrated aqueous ammonia-mediated thermochemical pretreatment (SCAA) would overcome the native recalcitrance of SB by enhancing cellulase accessibility of the embedded holocellulosic microfibrils. Results In this study, we designed an experiment considering response surface methodology (Taguchi method, L8 orthogonal array) to optimize sugar recovery from ammonia pretreated sugarcane bagasse (SB) by using the method of soaking in concentrated aqueous ammonia (SCAA-SB). Three independent variables: ammonia concentration, temperature and time, were selected at two levels with center point. The ammonia pretreated bagasse (SCAA-SB) was enzymatically hydrolysed by commercial enzymes (Celluclast 1.5 L and Novozym 188) using 15 FPU/g dry biomass and 17.5 Units of β-glucosidase/g dry biomass at 50°C, 150 rpm for 96 h. A maximum of 28.43 g/l reducing sugars corresponding to 0.57 g sugars/g pretreated bagasse was obtained from the SCAA-SB derived using a 20% v/v ammonia solution, at 70°C for 24 h after enzymatic hydrolysis. Among the tested parameters, pretreatment time showed the maximum influence (p value, 0.053282) while ammonia concentration showed the least influence (p value, 0.612552) on sugar recovery. The changes in the ultra-structure and crystallinity of native SCAA-SB and enzymatically hydrolysed SB were observed by scanning electron microscopy (SEM), x-ray diffraction (XRD) and solid-state 13C nuclear magnetic resonance (NMR) spectroscopy. The enzymatic hydrolysates and solid SCAA-SB were subjected to ethanol fermentation under separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) by Scheffersomyces (Pichia) stipitis NRRL Y-7124 respectively. Higher ethanol production (10.31 g/l and yield, 0.387 g/g) was obtained through SSF than SHF (3.83 g/l and yield, 0.289 g/g). Conclusions SCAA treatment showed marked lignin removal from SB thus improving the accessibility of cellulases towards holocellulose substrate as evidenced by efficient sugar release. The ultrastructure of SB after SCAA and enzymatic hydrolysis of holocellulose provided insights of the degradation process at the molecular level.

Genetic diversity and population structure of Musa accessions in ex situ conservation

Abstract Background Banana cultivars are mostly derived from hybridization between wild diploid subspecies of Musa acuminata (A genome) and M. balbisiana (B genome), and they exhibit various levels of ploidy and genomic constitution. The Embrapa ex situ Musa collection contains over 220 accessions, of which only a few have been genetically characterized. Knowledge regarding the genetic relationships and diversity between modern cultivars and wild relatives would assist in conservation and breeding strategies. Our objectives were to determine the genomic constitution based on Internal Transcribed Spacer (ITS) regions polymorphism and the ploidy of all accessions by flow cytometry and to investigate the population structure of the collection using Simple Sequence Repeat (SSR) loci as co-dominant markers based on Structure software, not previously performed in Musa. Results From the 221 accessions analyzed by flow cytometry, the correct ploidy was confirmed or established for 212 (95.9%), whereas digestion of the ITS region confirmed the genomic constitution of 209 (94.6%). Neighbor-joining clustering analysis derived from SSR binary data allowed the detection of two major groups, essentially distinguished by the presence or absence of the B genome, while subgroups were formed according to the genomic composition and commercial classification. The co-dominant nature of SSR was explored to analyze the structure of the population based on a Bayesian approach, detecting 21 subpopulations. Most of the subpopulations were in agreement with the clustering analysis. Conclusions The data generated by flow cytometry, ITS and SSR supported the hypothesis about the occurrence of homeologue recombination between A and B genomes, leading to discrepancies in the number of sets or portions from each parental genome. These phenomenons have been largely disregarded in the evolution of banana, as the “single-step domestication” hypothesis had long predominated. These findings will have an impact in future breeding approaches. Structure analysis enabled the efficient detection of ancestry of recently developed tetraploid hybrids by breeding programs, and for some triploids. However, for the main commercial subgroups, Structure appeared to be less efficient to detect the ancestry in diploid groups, possibly due to sampling restrictions. The possibility of inferring the membership among accessions to correct the effects of genetic structure opens possibilities for its use in marker-assisted selection by association mapping.

New insights regarding HCV-NS5A structure/function and indication of genotypic differences

Abstract Background HCV is prevalent throughout the world. It is a major cause of chronic liver disease. There is no effective vaccine and the most common therapy, based on Peginterferon, has a success rate of ~50%. The mechanisms underlying viral resistance have not been elucidated but it has been suggested that both host and virus contribute to therapy outcome. Non-structural 5A (NS5A) protein, a critical virus component, is involved in cellular and viral processes. Methods The present study analyzed structural and functional features of 345 sequences of HCV-NS5A genotypes 1 or 3, using in silico tools. Results There was residue type composition and secondary structure differences between the genotypes. In addition, second structural variance were statistical different for each response group in genotype 3. A motif search indicated conserved glycosylation, phosphorylation and myristoylation sites that could be important in structural stabilization and function. Furthermore, a highly conserved integrin ligation site was identified, and could be linked to nuclear forms of NS5A. ProtFun indicated NS5A to have diverse enzymatic and nonenzymatic activities, participating in a great range of cell functions, with statistical difference between genotypes. Conclusion This study presents new insights into the HCV-NS5A. It is the first study that using bioinformatics tools, suggests differences between genotypes and response to therapy that can be related to NS5A protein features. Therefore, it emphasizes the importance of using bioinformatics tools in viral studies. Data acquired herein will aid in clarifying the structure/function of this protein and in the development of antiviral agents.

Phylogeography of the neotropical Anopheles triannulatus complex (Diptera: Culicidae) supports deep structure and complex patterns

Abstract Background The molecular phylogenetic relationships and population structure of the species of the Anopheles triannulatus complex: Anopheles triannulatus s.s., Anopheles halophylus and the putative species Anopheles triannulatus C were investigated. Methods The mitochondrial COI gene, the nuclear white gene and rDNA ITS2 of samples that include the known geographic distribution of these taxa were analyzed. Phylogenetic analyses were performed using Bayesian inference, Maximum parsimony and Maximum likelihood approaches. Results Each data set analyzed septely yielded a different topology but none provided evidence for the seption of An. halophylus and An. triannulatus C, consistent with the hypothesis that the two are undergoing incipient speciation. The phylogenetic analyses of the white gene found three main clades, whereas the statistical parsimony network detected only a single metapopulation of Anopheles triannulatus s.l. Seven COI lineages were detected by phylogenetic and network analysis. In contrast, the network, but not the phylogenetic analyses, strongly supported three ITS2 groups. Combined data analyses provided the best resolution of the trees, with two major clades, Amazonian (clade I) and trans-Andean + Amazon Delta (clade II). Clade I consists of multiple subclades: An. halophylus + An. triannulatus C; trans-Andean Venezuela; central Amazonia + central Bolivia; Atlantic coastal lowland; and Amazon delta. Clade II includes three subclades: Panama; cis-Andean Colombia; and cis-Venezuela. The Amazon delta specimens are in both clades, likely indicating local sympatry. Spatial and molecular variance analyses detected nine groups, corroborating some of subclades obtained in the combined data analysis. Conclusion Combination of the three molecular markers provided the best resolution for differentiation within An. triannulatus s.s. and An. halophylus and C. The latest two species seem to be very closely related and the analyses performed were not conclusive regarding species differentiation. Further studies including new molecular markers would be desirable to solve this species status question. Besides, results of the study indicate a trans-Andean origin for An. triannulatus s.l. The potential implications for malaria epidemiology remain to be investigated.