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1.
Nat Commun ; 11(1): 303, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31949166

RESUMO

α-Dystroglycan (α-DG) is a highly-glycosylated surface membrane protein. Defects in the O-mannosyl glycan of α-DG cause dystroglycanopathy, a group of congenital muscular dystrophies. The core M3 O-mannosyl glycan contains tandem ribitol-phosphate (RboP), a characteristic feature first found in mammals. Fukutin and fukutin-related protein (FKRP), whose mutated genes underlie dystroglycanopathy, sequentially transfer RboP from cytidine diphosphate-ribitol (CDP-Rbo) to form a tandem RboP unit in the core M3 glycan. Here, we report a series of crystal structures of FKRP with and without donor (CDP-Rbo) and/or acceptor [RboP-(phospho-)core M3 peptide] substrates. FKRP has N-terminal stem and C-terminal catalytic domains, and forms a tetramer both in crystal and in solution. In the acceptor complex, the phosphate group of RboP is recognized by the catalytic domain of one subunit, and a phosphate group on O-mannose is recognized by the stem domain of another subunit. Structure-based functional studies confirmed that the dimeric structure is essential for FKRP enzymatic activity.


Assuntos
Distrofias Musculares/metabolismo , Açúcares de Nucleosídeo Difosfato/química , Açúcares de Nucleosídeo Difosfato/metabolismo , Pentosiltransferases/química , Pentosiltransferases/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Glicopeptídeos , Células HEK293 , Humanos , Modelos Moleculares , Distrofias Musculares/genética , Pentosiltransferases/genética , Fosfatos/metabolismo , Polissacarídeos/metabolismo , Conformação Proteica , Domínios Proteicos , Ribitol/metabolismo
2.
Biochim Biophys Acta Proteins Proteom ; 1868(1): 140292, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31676450

RESUMO

Enzymatic transglycosylation, a transfer of the carbohydrate moiety from one heterocyclic base to another, is catalyzed by nucleoside phosphorylases (NPs) and is being actively developed and applied for the synthesis of biologically important nucleosides. Here, we report an efficient one-step synthesis of 5-substitited pyrimidine ribonucleosides starting from 7-methylguanosine hydroiodide in the presence of nucleoside phosphorylases (NPs).


Assuntos
Proteínas de Bactérias/química , Escherichia coli/enzimologia , Pentosiltransferases/química , Ribonucleosídeos/química , Uridina/química , Proteínas de Bactérias/genética , Catálise , Glicosilação , Pentosiltransferases/genética , Proteínas Recombinantes/química
3.
Nat Commun ; 10(1): 4116, 2019 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-31511522

RESUMO

Damage-associated molecular patterns (DAMPs) are molecules that can be actively or passively released by injured tissues and that activate the immune system. Here we show that nicotinate phosphoribosyltransferase (NAPRT), detected by antibody-mediated assays and mass spectrometry, is an extracellular ligand for Toll-like receptor 4 (TLR4) and a critical mediator of inflammation, acting as a DAMP. Exposure of human and mouse macrophages to NAPRT activates the inflammasome and NF-κB for secretion of inflammatory cytokines. Furthermore, NAPRT enhances monocyte differentiation into macrophages by inducing macrophage colony-stimulating factor. These NAPRT-induced effects are independent of NAD-biosynthetic activity, but rely on NAPRT binding to TLR4. In line with our finding that NAPRT mediates endotoxin tolerance in vitro and in vivo, sera from patients with sepsis contain the highest levels of NAPRT, compared to patients with other chronic inflammatory conditions. Together, these data identify NAPRT as a endogenous ligand for TLR4 and a mediator of inflammation.


Assuntos
Espaço Extracelular/metabolismo , Inflamação/enzimologia , Pentosiltransferases/metabolismo , Receptor 4 Toll-Like/metabolismo , Diferenciação Celular , Líquido Extracelular/enzimologia , Humanos , Inflamação/genética , Inflamação/patologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/metabolismo , Monócitos/citologia , Células Mieloides/metabolismo , Nicotinamida Fosforribosiltransferase/química , Nicotinamida Fosforribosiltransferase/metabolismo , Pentosiltransferases/sangue , Pentosiltransferases/química , Ligação Proteica , Fatores de Risco , Sepse/sangue , Sepse/enzimologia
4.
Org Biomol Chem ; 17(34): 7891-7899, 2019 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-31397456

RESUMO

Insight into the catalytic mechanism of Lactobacillus leichmannii nucleoside 2'-deoxyribosyltransferase (LlNDT) has been gained by calculating a quantum mechanics-molecular mechanics (QM/MM) free-energy landscape of the reaction within the enzyme active site. Our results support an oxocarbenium species as the reaction intermediate and thus an SN1 reaction mechanism in this family of bacterial enzymes. Our mechanistic proposal is validated by comparing experimental kinetic data on the impact of the single amino acid replacements Tyr7, Glu98 and Met125 with Ala, Asp and Ala/norLeu, respectively, and accounts for the specificity shown by this enzyme on a non-natural substrate. This work broadens our understanding of enzymatic C-N bond cleavage and C-N bond formation.


Assuntos
Pentosiltransferases/química , Domínio Catalítico , Cinética , Lactobacillus leichmannii/enzimologia , Modelos Químicos , Simulação de Dinâmica Molecular , Estudo de Prova de Conceito , Conformação Proteica , Teoria Quântica , Termodinâmica
5.
Protoplasma ; 256(4): 1119-1132, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30941581

RESUMO

Xylosyltransferases (XylTs) play key roles in the biosynthesis of many different polysaccharides. These enzymes transfer D-xylose from UDP-xylose to substrate acceptors. In this study, we identified 30 XylTs from primary endosymbionts (green algae, red algae, and glaucophytes) and secondary or higher endosymbionts (brown algae, diatoms, Eustigmatophyceae, Pelagophyceae, and Cryptophyta). We performed comparative phylogenetic studies on key XylT subfamilies, and investigated the functional divergence of genes using RNA-Seq. Of the 30 XylTs, one ß-1,4-XylT IRX14-related, one ß-1,4 XylT IRX10L-related, and one xyloglucan 6-XylT 1-related gene were identified in the Charophyta, showing strong similarities to their land plant descendants. This implied the ancient occurrence of xylan and xyloglucan biosynthetic machineries in Charophyta. The other 27 XylTs were identified as UDP-D-xylose: L-fucose-α-1,3-D-XylT (FucXylT) type that specifically transferred D-xylose to fucose. We propose that FucXylTs originated from the last eukaryotic common ancestor, rather than being plant specific, because they are also distributed in Choanoflagellatea and Echinodermata. Considering the evidence from many aspects, we hypothesize that the FucXylTs likely participated in fucoidan biosynthesis in brown algae. We provide the first insights into the evolutionary history and functional divergence of FucXylT in algal biology.


Assuntos
Clorófitas/enzimologia , Pentosiltransferases/classificação , Pentosiltransferases/genética , Feófitas/enzimologia , Rodófitas/enzimologia , Carofíceas/genética , Clorófitas/genética , Embriófitas , Regulação da Expressão Gênica de Plantas , Pentosiltransferases/química , Pentosiltransferases/metabolismo , Feófitas/genética , Filogenia , Polissacarídeos , Conformação Proteica , Rodófitas/genética
6.
J Biosci Bioeng ; 128(1): 22-27, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30803783

RESUMO

Nucleoside deoxyribosyltransferase II (NDT) catalyzes the transglycosylation reaction of the 2'-deoxyribose moiety between purine and/or pyrimidine bases and has been widely used in the synthesis of nucleoside analogs. The high specificity of NDT for 2'-deoxyribose limits its applications. Because 2'C- and/or 3'C-modified nucleosides have been widely used as antiviral or antitumour agents, improving the activity of NDT towards these modified nucleosides by protein engineering is an area of interest to the pharmaceutical industry. NDT engineering is hindered by a lack of effective screening methods. This study developed a high-throughput screening system, which was established by nucleoside deoxyribosyltransferase II-cytidine deaminase co-expression, indophenol colorimetric assay and whole-cell catalysis. A high-throughput screening system for NDT was established for the first time. This system can be applied to detect NDT-specific activity for a variety of cytidine analogs with glycosyl and base modifications, such as 5-aza-2'-deoxycytidine, 2',3'-dideoxycytidine, cytosine-ß-d-arabinofuranoside. In this study, we adopted the semi-rational design of NDT and constructed a mutant library of NDT from Lactobacillus helveticus (LhNDT) by site-saturation mutagenesis. Over 600 mutants were screened, and a variant with up to a 5.2-fold higher conversion rate of 2',3'-dideoxyinosine was obtained.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Lactobacillus helveticus/genética , Proteínas Mutantes/isolamento & purificação , Pentosiltransferases/genética , Pentosiltransferases/isolamento & purificação , Pentosiltransferases/metabolismo , Catálise , Domínio Catalítico/genética , Ensaios Enzimáticos/métodos , Escherichia coli/enzimologia , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica , Mutagênese Sítio-Dirigida/métodos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Nucleosídeos , Pentosiltransferases/química , Engenharia de Proteínas/métodos , Purinas , Pirimidinas , Relação Estrutura-Atividade , Especificidade por Substrato/genética
7.
Biochemistry ; 58(7): 951-964, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30640434

RESUMO

Cobamides are coenzymes used by cells from all domains of life but made de novo by only some bacteria and archaea. The last steps of the cobamide biosynthetic pathway activate the corrin ring and the lower ligand base, condense the activated intermediates, and dephosphorylate the product prior to the release of the biologically active coenzyme. In bacteria, a phosphoribosyltransferase (PRTase) enyzme activates the base into its α-mononucleotide. The enzyme from Salmonella enterica ( SeCobT) has been extensively biochemically and structurally characterized. The crystal structure of the putative PRTase from the archaeum Methanocaldococcus jannaschii ( MjCobT) is known, but its function has not been validated. Here we report the in vivo and in vitro characterization of MjCobT. In vivo, in vitro, and phylogenetic data reported here show that MjCobT belongs to a new class of NaMN-dependent PRTases. We also show that the Synechococcus sp. WH7803 CobT protein has PRTase activity in vivo. Lastly, results of isothermal titration calorimetry and analytical ultracentrifugation analysis show that the biologically active form of MjCobT is a dimer, not a trimer, as suggested by its crystal structure.


Assuntos
Proteínas Arqueais/metabolismo , Proteínas de Bactérias/metabolismo , Cobamidas/biossíntese , Archaea/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cianobactérias/metabolismo , Concentração de Íons de Hidrogênio , Mathanococcus/enzimologia , Mathanococcus/genética , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Concentração Osmolar , Pentosiltransferases/química , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Fosfatos/química , Fosfatos/metabolismo , Filogenia , Compostos de Potássio/química , Compostos de Potássio/metabolismo , Salmonella enterica/genética , Salmonella enterica/metabolismo , Especificidade por Substrato
8.
PLoS One ; 13(10): e0206187, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30335828

RESUMO

The rare branched-chain sugar apiose, once thought to only be present in the plant kingdom, was found in two bacterial species: Geminicoccus roseus and Xanthomonas pisi. Glycans with apiose residues were detected in aqueous methanol-soluble fractions as well as in the insoluble pellet fraction of X. pisi. Genes encoding bacterial uridine diphosphate apiose (UDP-apiose) synthases (bUASs) were characterized in these bacterial species, but the enzyme(s) involved in the incorporation of the apiose into glycans remained unknown. In the X. pisi genome two genes flanking the XpUAS were annotated as hypothetical glycosyltransferase (GT) proteins. The first GT (here on named XpApiT) belongs to GT family 90 and has a Leloir type B fold and a putative lipopolysaccharide-modifying (LPS) domain. The second GT (here on XpXylT) belongs to GT family 2 and has a type A fold. The XpXylT and XpApiT genes were cloned and heterologously expressed in E. coli. Analysis of nucleotide sugar extracts from E. coli expressing XpXylT or XpApiT with UAS showed that recombinant XpApiT utilized UDP-apiose and XpXylT utilized UDP-xylose as substrate. Indirect activity assay (UDP-Glo) revealed that XpApiT is an apiosyltransferase (ApiT) able to specifically use UDP-apiose. Further support for the apiosyltransferase activity was demonstrated by in microbe co-expression of UAS and XpApiT in E. coli showing the utilization of UDP-apiose to generate an apioside detectable in the pellet fraction. This work provides evidence that X. pisi developed the ability to synthesize an apioside of indeterminate function; however, the evolution of the bacterial ApiT remains to be determined. From genetic and evolutionary perspectives, the apiose operon may provide a unique opportunity to examine how genomic changes reflect ecological adaptation during the divergence of a bacterial group.


Assuntos
Pentoses/metabolismo , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Xanthomonas/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Óperon , Pentosiltransferases/química , Filogenia , Plantas/microbiologia , Domínios Proteicos , Xanthomonas/genética
9.
J Comput Aided Mol Des ; 32(11): 1229-1245, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30196523

RESUMO

Here, we propose an in silico fragment-mapping method as a potential tool for fragment-based/structure-based drug discovery (FBDD/SBDD). For this method, we created a database named Canonical Subsite-Fragment DataBase (CSFDB) and developed a knowledge-based fragment-mapping program, Fsubsite. CSFDB consists of various pairs of subsite-fragments derived from X-ray crystal structures of known protein-ligand complexes. Using three-dimensional similarity-matching between subsites on one protein and another, Fsubsite compares the surface of a target protein with all subsites in CSFDB. When a local topography similar to the subsite is found on the surface, Fsubsite places a fragment combined with the subsite in CSFDB on the target protein. For validation purposes, we applied the method to the apo-structure of cyclin-dependent kinase 2 (CDK2) and identified four compounds containing three mapped fragments that existed in the list of known inhibitors of CDK2. Next, the utility of our fragment-mapping method for fragment-growing was examined on the complex structure of tRNA-guanine transglycosylase with a small ligand. Fsubsite mapped appropriate fragments on the same position as the binding ligand or in the vicinity of the ligand. Finally, a 3D-pharmacophore model was constructed from the fragments mapped on the apo-structure of heat shock protein 90-α (HSP90α). Then, 3D pharmacophore-based virtual screening was carried out using a commercially available compound database. The resultant hit compounds were very similar to a known ligand of HSP90α. As a result of these findings, this in silico fragment-mapping method seems to be a useful tool for computational FBDD and SBDD.


Assuntos
Simulação por Computador , Modelos Moleculares , Proteínas/química , Sítios de Ligação , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/química , Bases de Dados de Compostos Químicos , Descoberta de Drogas/métodos , Proteínas de Choque Térmico HSP90/química , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Bases de Conhecimento , Ligantes , Pentosiltransferases/química , Ligação Proteica , Conformação Proteica , Proteínas/antagonistas & inibidores , Software
10.
Glycobiology ; 28(11): 849-859, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30169771

RESUMO

In multicellular organisms, glycosylation regulates various developmental signaling pathways including the Notch pathway. One of the O-linked glycans added to epidermal growth factor-like (EGF) repeats in animal proteins including the Notch receptors is the xylose-xylose-glucose-O oligosaccharide. Drosophila glucoside xylosyltransferase (Gxylt) Shams negatively regulates Notch signaling in specific contexts. Since Shams adds the first xylose residue to O-glucose, its loss-of-function phenotype could be due to the loss of the first xylose, the second xylose or both. To examine the contribution of the second xylose residues to Drosophila Notch signaling, we have performed biochemical and genetic analysis on CG11388, which is the Drosophila homolog of human xyloside xylosyltransferase 1 (XXYLT1). Experiments in S2 cells indicated that similar to human XXYLT1, CG11388 can add the second xylose to xylose-glucose-O glycans. Flies lacking both copies of CG11388 (Xxylt) are viable and fertile and do not show gross phenotypes indicative of altered Notch signaling. However, genetic interaction experiments show that in sensitized genetic backgrounds with decreased or increased Notch pathway components, loss of Xxylt promotes Delta-mediated activation of Notch. Unexpectedly, we find that in such sensitized backgrounds, even loss of one copy of the fly Gxylt shams enhances Delta-mediated Notch activation. Taken together, these data indicate that while the first xylose plays a key role in tuning the Delta-mediated Notch signaling in Drosophila, the second xylose has a fine-tuning role only revealed in sensitized genetic backgrounds.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/metabolismo , Fator de Crescimento Epidérmico/química , Patrimônio Genético , Pentosiltransferases/química , Pentosiltransferases/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Proteínas de Drosophila/genética , Humanos , Pentosiltransferases/genética , Receptores Notch/genética , Transdução de Sinais/genética
11.
Biomolecules ; 8(3)2018 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-30149595

RESUMO

RNA modifications have been implicated in diverse and important roles in all kingdoms of life with over 100 of them present on tRNAs. A prominent modification at the wobble base of four tRNAs is the 7-deaza-guanine derivative queuine which substitutes the guanine at position 34. This exchange is catalyzed by members of the enzyme class of tRNA guanine transglycosylases (TGTs). These enzymes incorporate guanine substituents into tRNAAsp, tRNAAsn tRNAHis, and tRNATyr in all kingdoms of life. In contrast to the homodimeric bacterial TGT, the active eukaryotic TGT is a heterodimer in solution, comprised of a catalytic QTRT1 subunit and a noncatalytic QTRT2 subunit. Bacterial TGT enzymes, that incorporate a queuine precursor, have been identified or proposed as virulence factors for infections by pathogens in humans and therefore are valuable targets for drug design. To date no structure of a eukaryotic catalytic subunit is reported, and differences to its bacterial counterpart have to be deducted from sequence analysis and models. Here we report the first crystal structure of a eukaryotic QTRT1 subunit and compare it to known structures of the bacterial TGT and murine QTRT2. Furthermore, we were able to determine the crystal structure of QTRT1 in complex with the queuine substrate.


Assuntos
Domínio Catalítico , Pentosiltransferases/química , Apoenzimas/química , Apoenzimas/metabolismo , Cristalografia por Raios X , Guanina/metabolismo , Humanos , Modelos Moleculares , Pentosiltransferases/metabolismo
12.
Appl Microbiol Biotechnol ; 102(16): 6947-6957, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29872887

RESUMO

In our search for thermophilic and acid-tolerant nucleoside 2'-deoxyribosyltransferases (NDTs), we found a good candidate in an enzyme encoded by Chroococcidiopsis thermalis PCC 7203 (CtNDT). Biophysical and biochemical characterization revealed CtNDT as a homotetramer endowed with good activity and stability at both high temperatures (50-100 °C) and a wide range of pH values (from 3 to 7). CtNDT recognizes purine bases and their corresponding 2'-deoxynucleosides but is also proficient using cytosine and 2'-deoxycytidine as substrates. These unusual features preclude the strict classification of CtNDT as either a type I or a type II NDT and further suggest that this simple subdivision may need to be updated in the future. Our findings also hint at a possible link between oligomeric state and NDT's substrate specificity. Interestingly from a practical perspective, CtNDT displays high activity (80-100%) in the presence of several water-miscible co-solvents in a proportion of up to 20% and was successfully employed in the enzymatic production of several therapeutic nucleosides such as didanosine, vidarabine, and cytarabine.


Assuntos
Cianobactérias/enzimologia , Pentosiltransferases , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Temperatura Alta , Pentosiltransferases/biossíntese , Pentosiltransferases/química , Pentosiltransferases/genética , Pentosiltransferases/isolamento & purificação , Solventes/química , Especificidade por Substrato
13.
Biochemistry ; 57(26): 3953-3965, 2018 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-29862811

RESUMO

The bacterial enzyme tRNA-guanine transglycosylase (TGT) is involved in the biosynthesis of queuosine, a modified nucleoside present in the anticodon wobble position of tRNAHis, tRNATyr, tRNAAsp, and tRNAAsn. Although it forms a stable homodimer endowed with two active sites, it is, for steric reasons, able to bind and convert only one tRNA molecule at a time. In contrast, its mammalian counterpart constitutes a heterodimer consisting of a catalytic and a noncatalytic subunit, termed QTRT1 and QTRT2, respectively. Both subunits are homologous to the bacterial enzyme, yet only QTRT1 possesses all the residues required for substrate binding and catalysis. In mice, genetic inactivation of the TGT results in the uncontrolled oxidation of tetrahydrobiopterin and, accordingly, phenylketonuria-like symptoms. For this reason and because of the recent finding that mammalian TGT may be utilized for the treatment of multiple sclerosis, this enzyme is of potential medical relevance, rendering detailed knowledge of its biochemistry and structural architecture highly desirable. In this study, we performed the kinetic characterization of the murine enzyme, investigated potential quaternary structures of QTRT1 and QTRT2 via noncovalent mass spectrometry, and, finally, determined the crystal structure of the murine noncatalytic TGT subunit, QTRT2. In the crystal, QTRT2 is clearly present as a homodimer that is strikingly similar to that formed by bacterial TGT. In particular, a cluster of four aromatic residues within the interface of the bacterial TGT, which constitutes a "hot spot" for dimer stability, is present in a similar constellation in QTRT2.


Assuntos
Pentosiltransferases/química , Multimerização Proteica , Subunidades Proteicas/química , Animais , Cinética , Camundongos , Estrutura Quaternária de Proteína
14.
Angew Chem Int Ed Engl ; 57(32): 10085-10090, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-29927035

RESUMO

The enzyme tRNA-guanine transglycosylase, a target to fight Shigellosis, recognizes tRNA only as a homodimer and performs full nucleobase exchange at the wobble position. Active-site inhibitors block the enzyme function by competitively replacing tRNA. In solution, the wild-type homodimer dissociates only marginally, whereas mutated variants show substantial monomerization in solution. Surprisingly, one inhibitor transforms the protein into a twisted state, whereby one monomer unit rotates by approximately 130°. In this altered geometry, the enzyme is no longer capable of binding and processing tRNA. Three sugar-type inhibitors have been designed and synthesized, which bind to the protein in either the functionally competent or twisted inactive state. They crystallize with the enzyme side-by-side under identical conditions from the same crystallization well. Possibly, the twisted inactive form corresponds to a resting state of the enzyme, important for its functional regulation.


Assuntos
Pentosiltransferases/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Modelos Moleculares , Estrutura Molecular , Pentosiltransferases/antagonistas & inibidores , Pentosiltransferases/química
15.
Chemistry ; 24(39): 9957-9967, 2018 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-29939431

RESUMO

The intestinal disease shigellosis caused by Shigella bacteria affects over 120 million people annually. There is an urgent demand for new drugs as resistance against common antibiotics emerges. Bacterial tRNA-guanine transglycosylase (TGT) is a druggable target and controls the pathogenicity of Shigella flexneri. We report the synthesis of sugar-functionalized lin-benzoguanines addressing the ribose-33 pocket of TGT from Zymomonas mobilis. Ligand binding was analyzed by isothermal titration calorimetry and X-ray crystallography. Pocket occupancy was optimized by variation of size and protective groups of the sugars. The participation of a polycyclic water-cluster in the recognition of the sugar moiety was revealed. Acetonide-protected ribo- and psicofuranosyl derivatives are highly potent, benefiting from structural rigidity, good solubility, and metabolic stability. We conclude that sugar acetonides have a significant but not yet broadly recognized value in drug development.


Assuntos
Guanina/química , Pentosiltransferases/química , RNA de Transferência/química , Ribose/química , Açúcares/química , Zymomonas/química , Cristalografia por Raios X , Estrutura Molecular , Pentosiltransferases/metabolismo , Ligação Proteica , Solventes
16.
Structure ; 26(6): 801-809.e3, 2018 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-29681470

RESUMO

Proteoglycans (PGs) are essential components of the animal extracellular matrix and are required for cell adhesion, migration, signaling, and immune function. PGs are composed of a core protein and long glycosaminoglycan (GAG) chains, which often specify PG function. GAG biosynthesis is initiated by peptide O-xylosyltransferases, which transfer xylose onto selected serine residues in the core proteins. We have determined crystal structures of human xylosyltransferase 1 (XT1) in complex with the sugar donor, UDP-xylose, and various acceptor peptides. The structures reveal unique active-site features that, in conjunction with functional experiments, explain the substrate specificity of XT1. A constriction within the peptide binding cleft requires the acceptor serine to be followed by glycine or alanine. The remainder of the cleft can accommodate a wide variety of sequences, but with a general preference for acidic residues. These findings provide a framework for understanding the selectivity of GAG attachment.


Assuntos
Glicosaminoglicanos/biossíntese , Pentosiltransferases/química , Pentosiltransferases/metabolismo , Uridina Difosfato Xilose/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Glicosilação , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Serina/química , Especificidade por Substrato
17.
Acta Crystallogr F Struct Biol Commun ; 74(Pt 3): 161-165, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29497020

RESUMO

Tuberculosis (TB) continues to remain a leading cause of death globally. Of particular concern is the emergence and rise in incidence of multidrug-resistant and extremely drug-resistant cases of TB. To counter this threat, it is important to explore alternative therapies, including phage therapy. Phage BTCU-1 specifically infects Mycobacterium spp. and kills the majority of them. Intriguingly, many proteins from the phage do not share high amino-acid sequence identity with proteins from species other than phages. Here, the expression, purification and crystallization of one such protein, a putative phosphoribosyl transferase from phage BTCU-1, is reported. The crystals belonged to space group C2221, with unit-cell parameters a = 59.71, b = 64.42, c = 65.32 Å, α = ß = γ = 90°. The crystals diffracted X-rays to 2.2 Šresolution.


Assuntos
Micobacteriófagos/enzimologia , Pentosiltransferases/química , Pentosiltransferases/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Pentosiltransferases/isolamento & purificação , Conformação Proteica , Homologia de Sequência , Proteínas Virais/isolamento & purificação
18.
PLoS Negl Trop Dis ; 12(2): e0006301, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29481567

RESUMO

Due to toxicity and compliance issues and the emergence of resistance to current medications new drugs for the treatment of Human African Trypanosomiasis are needed. A potential approach to developing novel anti-trypanosomal drugs is by inhibition of the 6-oxopurine salvage pathways which synthesise the nucleoside monophosphates required for DNA/RNA production. This is in view of the fact that trypanosomes lack the machinery for de novo synthesis of the purine ring. To provide validation for this approach as a drug target, we have RNAi silenced the three 6-oxopurine phosphoribosyltransferase (PRTase) isoforms in the infectious stage of Trypanosoma brucei demonstrating that the combined activity of these enzymes is critical for the parasites' viability. Furthermore, we have determined crystal structures of two of these isoforms in complex with several acyclic nucleoside phosphonates (ANPs), a class of compound previously shown to inhibit 6-oxopurine PRTases from several species including Plasmodium falciparum. The most potent of these compounds have Ki values as low as 60 nM, and IC50 values in cell based assays as low as 4 µM. This data provides a solid platform for further investigations into the use of this pathway as a target for anti-trypanosomal drug discovery.


Assuntos
Inibidores Enzimáticos/farmacologia , Redes e Vias Metabólicas/efeitos dos fármacos , Purinonas/metabolismo , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/metabolismo , Domínio Catalítico , Descoberta de Drogas , Inibidores Enzimáticos/química , Humanos , Hipoxantina Fosforribosiltransferase/antagonistas & inibidores , Hipoxantina Fosforribosiltransferase/química , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Modelos Moleculares , Pentosiltransferases/antagonistas & inibidores , Pentosiltransferases/química , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Interferência de RNA , Tripanossomicidas/química , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genética
19.
New Phytol ; 218(3): 1049-1060, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29460505

RESUMO

Insights into the evolution of plant cell walls have important implications for comprehending these diverse and abundant biological structures. In order to understand the evolving structure-function relationships of the plant cell wall, it is imperative to trace the origin of its different components. The present study is focused on plant 1,4-ß-xylan, tracing its evolutionary origin by genome and transcriptome mining followed by phylogenetic analysis, utilizing a large selection of plants and algae. It substantiates the findings by heterologous expression and biochemical characterization of a charophyte alga xylan synthase. Of the 12 known gene classes involved in 1,4-ß-xylan formation, XYS1/IRX10 in plants, IRX7, IRX8, IRX9, IRX14 and GUX occurred for the first time in charophyte algae. An XYS1/IRX10 ortholog from Klebsormidium flaccidum, designated K. flaccidumXYLAN SYNTHASE-1 (KfXYS1), possesses 1,4-ß-xylan synthase activity, and 1,4-ß-xylan occurs in the K. flaccidum cell wall. These data suggest that plant 1,4-ß-xylan originated in charophytes and shed light on the origin of one of the key cell wall innovations to occur in charophyte algae, facilitating terrestrialization and emergence of polysaccharide-based plant cell walls.


Assuntos
Parede Celular/metabolismo , Carofíceas/enzimologia , Pentosiltransferases/metabolismo , Células Vegetais/metabolismo , Motivos de Aminoácidos , Vias Biossintéticas , Carofíceas/genética , Evolução Molecular , Células HEK293 , Humanos , Pentosiltransferases/química , Filogenia
20.
Plant Cell Physiol ; 59(3): 554-565, 2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-29325159

RESUMO

Xylan is a major hemicellulose in both primary and secondary walls of grass species. It consists of a linear backbone of ß-1,4-linked xylosyl residues that are often substituted with monosaccharides and disaccharides. Xylosyl substitutions directly on the xylan backbone have not been reported in grass species, and genes responsible for xylan substitutions in grass species have not been well elucidated. Here, we report functional characterization of a rice (Oryza sativa) GT61 glycosyltransferase, XYXT1 (xylan xylosyltransferase1), for its role in xylan substitutions. XYXT1 was found to be ubiquitously expressed in different rice organs and its encoded protein was targeted to the Golgi, the site for xylan biosynthesis. When expressed in the Arabidopsis gux1/2/3 triple mutant, in which xylan was completely devoid of sugar substitutions, XYXT1 was able to add xylosyl side chains onto xylan. Glycosyl linkage analysis and comprehensive structural characterization of xylooligomers generated by xylanase digestion of xylan from transgenic Arabidopsis plants expressing XYXT1 revealed that the side chain xylosyl residues were directly attached to the xylan backbone at O-2, a substituent not present in wild-type Arabidopsis xylan. XYXT1 was unable to add xylosyl residues onto the arabinosyl side chains of xylan when it was co-expressed with OsXAT2 (Oryza sativa xylan arabinosyltransferase2) in the gux1/2/3 triple mutant. Furthermore, we showed that recombinant XYXT1 possessed an activity transferring xylosyl side chains onto xylooligomer acceptors, whereas recombinant OsXAT2 catalyzed the addition of arabinosyl side chains onto xylooligomer acceptors. Our findings from both an in vivo gain-of-function study and an in vitro recombinant protein activity assay demonstrate that XYXT1 is a novel ß-1,2-xylosyltransferase mediating the addition of xylosyl side chains onto xylan.


Assuntos
Oryza/enzimologia , Pentosiltransferases/metabolismo , Xilanos/metabolismo , Xilose/metabolismo , Arabidopsis/genética , Biocatálise , Regulação da Expressão Gênica de Plantas , Mutação/genética , Oryza/genética , Pentosiltransferases/química , Filogenia , Espectroscopia de Prótons por Ressonância Magnética , Proteínas Recombinantes/metabolismo
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