I04-1-Macromolecular Crystallography (fixed wavelength)
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Juliana Roverta
Torini
,
Adriano
De Freitas Fernandes
,
Vitor Hugo
Balasco Serrao
,
Larissa
Romanello
,
Louise
Bird
,
Joanne E.
Nettleship
,
Raymond J.
Owens
,
Jose
Brandao-neto
,
Ana Eliza
Zeraik
,
Ricardo
Demarco
,
Humberto
D'muniz Pereira
Diamond Proposal Number(s):
[5073, 14493]
Abstract: Nucleoside diphosphate kinases (NDPKs) are crucial to keep the high triphosphate nucleotide levels in the biological process. The enzymatic mechanism has been extensively described; however, the structural characteristics and kinetic parameters have never been fully determined. In Schistosoma mansoni, NDPK (SmNDPK) is directly involved in the pyrimidine and purine salvage pathways, being essential for nucleotide metabolism. The SmNDPK enzymatic activity is the highest of the known purine metabolisms when compared to the mammalian NDPKs, suggesting the importance of this enzyme in the worm metabolism. Here, we report the recombinant expression of SmNDPK that resulted in 1.7 and 1.9 Å apo-form structure in different space-groups, as well as the 2.1 Å SmNDPK.ADP complex. The binding and kinetic assays reveal the ATP-dependence for enzyme activation. Moreover, in situ hybridization showed that SmNDPK transcripts are found in reproductive organs and in the esophagus gland of adult worms, which can be intrinsically related with the oviposition and digestive processes. These results will help us fully understand the crucial participation of this enzyme in Schistosoma mansoni and its importance for the pathology of the disease.
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Jul 2019
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14496]
Abstract: Septins are GTP-binding proteins that will often spontaneously assemble into filaments. In some species, particularly budding yeast, it is well known that these are capable of associating with membranes in order to fulfill their cellular role as a component of the cytoskeleton. Different from other human septins, SEPT7 appears to be unique in that it is an essential component of all hetero-oligomeric complexes described to date. As a step towards understanding the molecular basis of filament assembly, here we present two high-resolution structures of the SEPT7 GTPase domain complexed with GDP. One of these reveals a previously unreported coordination for the magnesium ion involving four water molecules and only a tenuous connection to the protein. The higher resolution structures provide unambiguous insight into the interactions at the G-interface where a structural motif based on an antiparallel β-bridge allows for the rationalization of why some septins show nucleotide-dependent β-strand slippage and others do not.
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Apr 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Larissa
Romanello
,
Ana Eliza
Zeraik
,
Adriano
De Freitas Fernandes
,
Juliana Roberta
Torini
,
Louise E.
Bird
,
Joanne E.
Nettleship
,
Heather
Rada
,
Yamini
Reddivari
,
Ray J.
Owens
,
Vitor Hugo Balasco
Serrão
,
Ricardo
Demarco
,
Jose
Brandao-neto
,
Humberto
D'muniz Pereira
Diamond Proposal Number(s):
[5073]
Abstract: Schistosoma mansoni, the parasite responsible for schistosomiasis, lacks the “de novo” purine biosynthetic pathway and depends entirely on the purine salvage pathway for the supply of purines. Numerous reports of praziquantel resistance have been described, as well as stimulated efforts to develop new drugs against schistosomiasis. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme of the purine salvage pathway. Here, we describe a crystallographic structure of the S. mansoni HPGRT-1 (SmHGPRT), complexed with IMP at a resolution of 2.8Ǻ. Four substitutions were identified in the region of the active site between SmHGPRT-1 and human HGPRT. We also present data from RNA-Seq and WISH, suggesting that some isoforms of HGPRT might be involved in the process related to sexual maturation and reproduction in worms; furthermore, its enzymatic assays show that the isoform SmHGPRT-3 does not present the same catalytic efficiency as other isoforms. Finally, although other studies have previously suggested this enzyme as a potential antischistosomal chemotherapy target, the kinetics parameters reveal the impossibility to use SmHGPRT as an efficient chemotherapeutic target.
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Feb 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Juliana Roberta
Torini
,
Larissa
Romanello
,
Fernanda Aparecida Heleno
Batista
,
Vitor Hugo Balasco
Serrao
,
Muhamamd
Faheem
,
Ana Eliza
Zeraik
,
Louise
Bird
,
Joanne E.
Nettleship
,
Yamini
Reddivari
,
Ray
Owens
,
Ricardo
Demarco
,
Júlio César
Borges
,
Jose
Brandao-neto
,
Humberto
D'muniz Pereira
Open Access
Abstract: Purine nucleoside phosphorylases (PNPs) play an important role in the blood fluke parasite Schistosoma mansoni as a key enzyme of the purine salvage pathway. Here we present the structural and kinetic characterization of a new PNP isoform from S. mansoni, SmPNP2. Thermofluorescence screening of different ligands suggested cytidine and cytosine are potential ligands. The binding of cytosine and cytidine were confirmed by isothermal titration calorimetry, with a KD of 27 μM for cytosine, and a KM of 76.3 μM for cytidine. SmPNP2 also displays catalytic activity against inosine and adenosine, making it the first described PNP with robust catalytic activity towards both pyrimidines and purines. Crystal structures of SmPNP2 with different ligands were obtained and comparison of these structures with the previously described S. mansoni PNP (SmPNP1) provided clues for the unique capacity of SmPNP2 to bind pyrimidines. When compared with the structure of SmPNP1, substitutions in the vicinity of SmPNP2 active site alter the architecture of the nucleoside base binding site thus permitting an alternative binding mode for nucleosides, with a 180° rotation from the canonical binding mode. The remarkable plasticity of this binding site enhances our understanding of the correlation between structure and nucleotide selectivity, thus suggesting new ways to analyse PNP activity.
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Sep 2018
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Abstract: One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders Thermococcales, Methanosarcinales, and Methanococcales, as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts. We found that a major shift in function from a bifunctional ancestor that could phosphorylate either glucose or fructose 6-phosphate (fructose-6P) as a substrate to a fructose-6P-specific enzyme was started by a single amino acid substitution resulting in negative selection with a ground-state mode against glucose and a subsequent 1,600-fold change in specificity of the ancestral protein. This change rendered the residual phosphorylation of glucose a promiscuous and physiologically irrelevant activity, highlighting how promiscuity may be an evolutionary vestige of ancestral enzyme activities, which have been eliminated over time. We also could reconstruct the evolutionary history of substrate utilization by using an evolutionary model of discrete binary characters, indicating that substrate uses can be discretely lost or acquired during enzyme evolution. These findings exemplify how negative selection and subtle enzyme changes can lead to major evolutionary shifts in function, which can subsequently generate important adaptive advantages, for example, in improving glycolytic efficiency in Thermococcales.
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Jul 2017
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Andressa P. A.
Pinto
,
Humberto M.
Pereira
,
Ana E.
Zeraik
,
Heloisa
Ciol
,
Frederico M.
Ferreira
,
Jose
Brandao-neto
,
Ricardo
Demarco
,
Marcos V. A. S.
Navarro
,
Cristina
Risi
,
Vitold E.
Galkin
,
Richard C.
Garratt
,
Ana P. U.
Araujo
Diamond Proposal Number(s):
[5073]
Abstract: Septins are filament−forming GTP−binding proteins involved in many essential cellular events related to cytoskeletal dynamics and maintenance. Septins can self−assemble into hetero−complexes, which polymerize into highly organized, cell membrane−interacting filaments. The number of septin genes varies among organisms, and although their structure and function have been thoroughly studied in opisthokonts (including animals and fungi), no structural studies have been reported for other organisms. This makes the single septin from Chlamydomonas (CrSEPT) a particularly attractive model for investigating whether functional homopolymeric septin filaments also exist. CrSEPT was detected at the base of the flagella in Chlamydomonas, suggesting that CrSEPT is involved in the formation of a membrane diffusion barrier. Using transmission electron microscopy, we observed that recombinant CrSEPT forms long filaments with dimensions comparable to those of the canonical structure described for opisthokonts. The GTP−binding domain of CrSEPT purified as a nucleotide−free monomer that hydrolyzes GTP and readily binds its analog GTPγS. We also found that on nucleotide binding, CrSEPT forms dimers that are stabilized by an interface involving the ligand (G−interface). Across this interface, one monomer supplied a catalytic arginine to the opposing subunit, greatly accelerating the rate of GTP hydrolysis. This is the first report of an arginine finger observed in a septin and suggests that CrSEPT may act as its own GTP−activating protein (GAP). The finger is conserved in all algal septin sequences, suggesting a possible correlation between the ability to form homo−polymeric filaments and the accelerated rate of hydrolysis which it provides.
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May 2017
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Abstract: The parasite Schistosoma mansoni possesses all pathways for pyrimidine biosynthesis, in which dihydrofolate reductase (DHFR), thymidylate cycle participants, is essential for nucleotide metabolism to obtain energy and structural nucleic acids. Thus, DHFRs have been widely suggested as therapeutic targets for the treatment of infectious diseases. In this study, we expressed recombinantSmDHFR in a heterologous manner to obtain structural, biochemical and kinetic information. X-ray diffraction of recombinantSmDHFR at 1.95 Å resolution showed that the structure exhibited the canonical DHFR fold. Isothermal titration calorimetry was used to determine the kinetic constants for NADP+ and dihydrofolate. Moreover, inhibition assays were performed using the commercial folate analogs methotrexate and aminopterin; these analogs are recognized as folate competitors and are used as chemotherapeutic agents in cancer and autoimmune diseases. This study provides information that may prove useful for the future discovery of novel drugs and for understanding these metabolic steps from this pathway of S. mansoni, thus aiding in our understanding of the function of these essential pathways for parasite metabolism.
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Mar 2017
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I02-Macromolecular Crystallography
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Larissa
Romanello
,
Vitor Hugo Balasco
Serrao
,
Juliana Roberta Torini
De Souza
,
Louise E.
Bird
,
Joanne E.
Nettleship
,
Heather
Rada
,
Yamini
Reddivari
,
Ray J.
Owens
,
Ricardo
De Marco
,
Jose
Brandao-neto
,
Humberto
D' Muniz Pereira
Diamond Proposal Number(s):
[5073, 5894]
Abstract: Schistosoma mansoni is the parasite responsible for schistosomiasis, a disease that affects about 218 million people worldwide. Currently, both direct treatment and disease control initiatives rely on chemotherapy using a single drug, praziquantel. Concerns over the possibility of resistance developing to praziquantel, have stimulated efforts to develop new drugs for the treatment of schistosomiasis. Schistosomes do not have the de novo purine biosynthetic pathway, and instead depend entirely on the purine salvage pathway to supply its need for purines. The purine salvage pathway has been reported as a potential target for developing new drugs against schistosomiasis. Adenylosuccinate lyase (SmADSL) is an enzyme in this pathway, which cleaves adenylosuccinate (ADS) into adenosine 5'-monophosphate (AMP) and fumarate. SmADSL kinetic characterization was performed by isothermal titration calorimetry (ITC) using both ADS and SAICAR as substrates. Structures of SmADSL in Apo form and in complex with AMP were elucidated by x-ray crystallography revealing a highly conserved tetrameric structure required for their function since the active sites is formed from residues of three different subunits. The active sites are also highly conserved between species and it is difficult to identify a potent species-specific inhibitor for the development of new therapeutic agents. In contrast, several mutagenesis studies have demonstrated the importance of dimeric interface residues in the stability of the quaternary structure of the enzyme. The lower conservation of these residues between SmADSL and human ADSL could be used to lead the development of anti-schistosomiasis drugs based on disruption of subunit interfaces. These structures and kinetics data add another layer of information to Schistosoma mansoni purine salvage pathway.
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Mar 2017
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I04-Macromolecular Crystallography
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Patrícia G. Castro
Landim
,
Tuana O.
Correia
,
Fredy D. A.
Silva
,
Denise R.
Nepomuceno
,
Helen P. S.
Costa
,
Humberto
Pereira
,
Marina D. P.
Lobo
,
Frederico B. M. B.
Moreno
,
Jose
Brandao-neto
,
Suelen C.
Medeiros
,
Ilka M.
Vasconcelos
,
José T.a.
Oliveira
,
Bruno L.
Sousa
,
Ito L.
Barroso-neto
,
Valder N.
Freire
,
Cristina P. S.
Carvalho
,
Ana C. O.
Monteiro-moreira
,
Thalles B.
Grangeiro
Diamond Proposal Number(s):
[6397]
Abstract: A cowpea class I chitinase (VuChiI) was expressed in the methylotrophic yeast P. pastoris. The recombinant protein was secreted into the culture medium and purified by affinity chromatography on a chitin matrix. The purified chitinase migrated on SDS-polyacrylamide gel electrophoresis as two closely-related bands with apparent molecular masses of 34 and 37 kDa. The identity of these bands as VuChiI was demonstrated by mass spectrometry analysis of tryptic peptides and N-terminal amino acid sequencing. The recombinant chitinase was able to hydrolyze colloidal chitin but did not exhibit enzymatic activity toward synthetic substrates. The highest hydrolytic activity of the cowpea chitinase toward colloidal chitin was observed at pH 5.0. Furthermore, most VuChiI activity (approximately 92%) was retained after heating to 50 °C for 30 min, whereas treatment with 5 mM Cu2+ caused a reduction of 67% in the enzyme's chitinolytic activity. The recombinant protein had antifungal activity as revealed by its ability to inhibit the spore germination and mycelial growth of Penicillium herquei. The three-dimensional structure of VuChiI was resolved at a resolution of 1.55 Å by molecular replacement. The refined model had 245 amino acid residues and 381 water molecules, and the final R-factor and Rfree values were 14.78 and 17.22%, respectively. The catalytic domain of VuChiI adopts an α-helix-rich fold, stabilized by 3 disulfide bridges and possessing a wide catalytic cleft. Analysis of the crystallographic model and molecular docking calculations using chito-oligosaccharides provided evidences about the VuChiI residues involved in sugar binding and catalysis, and a possible mechanism of antifungal action is suggested.
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Jan 2017
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[11175]
Abstract: Citrus canker, caused by bacteria Xanthomonas citri subsp. citri, can affect all economically important varieties of citrus. Studying Xanthomonas genes related to the invasive capacity may improve the knowledge on how this works and ultimately use the information to avoid the disease. Some annotated genes from Xanthomonas citri subsp. citri published genome are addressed to an interesting class of genes named “pathogenicity, virulence and adaptation”. One of them is xanA, which encodes a predicted phosphoglucomutase. Phosphoglucomutases are ubiquitous enzymes among the living kingdoms that play roles in carbohydrate metabolism, catalyzing the reversible conversion of 1- to 6-phosphoglucose. In Xanthomonas, phosphoglucomutase activity is required to synthesize precursors of the pathogenesis-related polysaccharide xanthan. In this work, a characterization of this gene product is presented by structural and functional studies. Molecular cloning was used for heterologous expression and deletion of xanA. A Michaelis-Menten kinetics model was obtained using the recombinant protein. The protein structure was also determined by X-ray diffraction on the recombinant enzyme substrate-free, bound to glucose-1,6-biphosphate and to glucose-1-phosphate. Deletion of xanA was done with a suicide plasmid construct and the obtained mutant was tested for pathogenic capacity. This study is the first describing the properties of the Xanthomonas citri subsp. citri phosphoglucomutase.
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Dec 2016
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