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1.
J Biol Chem ; 295(10): 2932-2947, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-31980461

RESUMO

The δ isozyme of diacylglycerol kinase (DGKδ) plays critical roles in lipid signaling by converting diacylglycerol (DG) to phosphatidic acid (PA). We previously demonstrated that DGKδ preferably phosphorylates palmitic acid (16:0)- and/or palmitoleic acid (16:1)-containing DG molecular species, but not arachidonic acid (20:4)-containing DG species, which are recognized as DGK substrates derived from phosphatidylinositol turnover, in high glucose-stimulated myoblasts. However, little is known about the origin of these DG molecular species. DGKδ and two DG-generating enzymes, sphingomyelin synthase (SMS) 1 and SMS-related protein (SMSr), contain a sterile α motif domain (SAMD). In this study, we found that SMSr-SAMD, but not SMS1-SAMD, co-immunoprecipitates with DGKδ-SAMD. Full-length DGKδ co-precipitated with full-length SMSr more strongly than with SMS1. However, SAMD-deleted variants of SMSr and DGKδ interacted only weakly with full-length DGKδ and SMSr, respectively. These results strongly suggested that DGKδ interacts with SMSr through their respective SAMDs. To determine the functional outcomes of the relationship between DGKδ and SMSr, we used LC-MS/MS to investigate whether overexpression of DGKδ and/or SMSr in COS-7 cells alters the levels of PA species. We found that SMSr overexpression significantly enhances the production of 16:0- or 16:1-containing PA species such as 14:0/16:0-, 16:0/16:0-, 16:0/18:1-, and/or 16:1/18:1-PA in DGKδ-overexpressing COS-7 cells. Moreover, SMSr enhanced DGKδ activity via their SAMDs in vitro Taken together, these results strongly suggest that SMSr is a candidate DG-providing enzyme upstream of DGKδ and that the two enzymes represent a new pathway independent of phosphatidylinositol turnover.


Assuntos
Diacilglicerol Quinase/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Cromatografia Líquida de Alta Pressão , Diacilglicerol Quinase/química , Diacilglicerol Quinase/genética , Humanos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Alinhamento de Sequência , Motivo Estéril alfa , Espectrometria de Massas em Tandem , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/genética
2.
PLoS One ; 14(12): e0226579, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31856184

RESUMO

OBJECTIVE: Fluorescent labeling of specific cell-surface proteins enables a manifold of techniques to study their function in health and disease. A frequently cited family of methods employs phosphopantetheinyl transferases (PPTases) to attach probes, provided as conjugates of Coenzyme A. This method appears attractive, as only short peptide tags genetically fused to the protein of interest are needed as conjugation sites. Here, we describe observations we made when evaluating such protocols for delicate single-molecule applications where we require a particular combination of dyes, low background binding or low labeling of other proteins, and a high degree of labeling. RESULTS: When we tested a PPTase-acceptor peptide couple with several experimental protocols and various CoA conjugates for labeling of a protein on the cell surface, we noticed substantial non-specific labeling. For the first time, we provide here a quantification of the non-specific fraction of the signals obtained using appropriate controls. We further present evidence that this background is due to CoA-dye conjugates entering the cell, where they may be covalently attached to endogenous proteins. However, when studying cell-surface proteins, most fluorescent readouts require that labeling is strictly limited to the protein of interest located at the cell surface. While such data have so far been missing in the literature, they suggest that for applications where labeling of unwanted molecules would affect the conclusions, researchers need to be aware of this potential non-specificity of PPTase methods when selecting a labeling strategy. We show, again by quantitative comparison, that the HaloTag is a viable alternative.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Corantes Fluorescentes/química , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Coenzima A/química , Coenzima A/metabolismo , Células HEK293 , Humanos , Especificidade por Substrato
3.
Arch Biochem Biophys ; 675: 108111, 2019 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-31563509

RESUMO

Long-chain polyprenol phosphates feature in membrane-associated glycoconjugate biosynthesis pathways across domains of life. These unique amphiphilic molecules are best known as substrates of polytopic membrane proteins, including polyprenol-phosphate phosphoglycosyl and glycosyl transferases, and as components of more complex substrates. The linear polyprenols are constrained by double bond geometry and lend themselves well to interactions with polytopic membrane proteins, in which multiple transmembrane helices form a rich landscape for interactions. Recently, a new superfamily of monotopic phosphoglycosyl transferase enzymes has been identified that interacts with polyprenol phosphate substrates via a single reentrant membrane helix. Intriguingly, despite the dramatic differences in their membrane-interaction domains, both polytopic and monotopic enzymes similarly favor a unique cis/trans geometry in their polyprenol phosphate substrates. Herein, we present a multipronged biochemical and biophysical study of PglC, a monotopic phosphoglycosyl transferase that catalyzes the first membrane-committed step in N-linked glycoprotein biosynthesis in Campylobacter jejuni. We probe the significance of polyprenol phosphate geometry both in mediating substrate binding to PglC and in modulating the local membrane environment. Geometry is found to be important for binding to PglC; a conserved proline residue in the reentrant membrane helix is determined to drive polyprenol phosphate recognition and specificity. Pyrene fluorescence studies show that polyprenol phosphates at physiologically-relevant levels increase the disorder of the local lipid bilayer; however, this effect is confined to polyprenol phosphates with specific isoprene geometries. The molecular insights from this study may shed new light on the interactions of polyprenol phosphates with diverse membrane-associated proteins in glycoconjugate biosynthesis.


Assuntos
Poliprenois/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Campylobacter jejuni/crescimento & desenvolvimento , Campylobacter jejuni/metabolismo , Sequência Conservada , Fluidez de Membrana , Lipídeos de Membrana/metabolismo , Ligação Proteica , Conformação Proteica , Especificidade por Substrato , Transferases (Outros Grupos de Fosfato Substituídos)/química
4.
Mol Biotechnol ; 61(11): 836-851, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31482467

RESUMO

Phosphopantetheinyl transferases are of tremendous enthusiasm inferable from their fundamental parts in activating polyketide, fatty acid, and non-ribosomal peptide synthetase enzymes and additionally an increasing number of biotechnological applications. The present study reports the identification of sfp gene from the Paenibacillus sp. D9, which encompasses 693 bp encoding a 230-amino acid protein with a molecular weight of 25.3 kDa. The amino acid sequence Paenibacillus sp. D9 Sfp revealed more than 90% sequence identity to other Sfp proteins from other Paenibacillus. The sfp gene was cloned and recovered efficiently using affinity chromatography with maximal specific phosphopantetheinyl transferase activity at an optimal pH of 8.0 and temperature of 30 °C. The enzyme also exhibited stability under a wide-ranging pH and temperature. The presence of Zn2+, Cu2+, and Fe2+ ions improved the enzymatic activity, while other metals such as Ni2+, Co2+, and Mg2+ had inhibitory effects. The introduction of EDTA also displayed no inhibition. Kinetic parameters were obtained having values of 4.52 mg/mL, 35.33 U/mg, 3.64 s-1, and 0.104 mM-1 s-1 for Km, Vmax, kcat, and kcat/Km, respectively. The biosurfactant synthesized by the recombinant BioSp was found to be surface active, reducing the surface tension to 33.7 mN/m on the glucose substrate after 5 days of incubation at 37 °C. The recombinant Escherichia coli strain also exhibited an improvement in biosurfactant yield (1.11 g/L) when contrasted with 0.52 g/L from Paenibacillus sp. D9. High esterase activity of 2.55 IU/mL using p-nitrophenyl acetate was observed on the recombinant strain, as the protein connected with the release of the biosurfactant was observed to be an esterase. The characteristics of improved biosurfactant and esterase synthesis by hyper-producing recombinant strain possess numerous values from biotechnology standpoint.


Assuntos
Proteínas de Bactérias/metabolismo , Lipopeptídeos/biossíntese , Paenibacillus/enzimologia , Tensoativos/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Sequência de Aminoácidos/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Biotecnologia , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Esterases/metabolismo , Cinética , Metais Pesados/metabolismo , Peptídeo Sintases/metabolismo , Filogenia , Tensoativos/química , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/isolamento & purificação
5.
Methods Mol Biol ; 2012: 237-278, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31161512

RESUMO

4'-Phosphopantetheinyl transferases (PPTases) have been employed by researchers as versatile biocatalysts for the site-specific modification of numerous protein targets with structurally diverse molecules. Here we describe the use of these enzymes for the production of homogeneous antibody-drug conjugates (ADCs), which have garnered much attention as innovative anticancer drugs. The exceptionally broad substrate tolerance of PPTases allows for one-step and two-step conjugation strategies for site-specific ADC synthesis. While one-step conjugation involves direct coupling of a drug molecule to an antibody, two-step conjugation provides increased flexibility and efficiency of the conjugation process by first attaching a bioorthogonal chemical handle that is then used for drug molecule attachment in a second step. The aim of this chapter is to outline detailed protocols for both labeling procedures, as well as to provide guidance on enzyme and substrate preparation.


Assuntos
Anticorpos/química , Proteínas de Bactérias/química , Imunoconjugados/química , Transferases (Outros Grupos de Fosfato Substituídos)/química , Antineoplásicos/química , Catálise , Estrutura Molecular , Proteínas Recombinantes , Relação Estrutura-Atividade
6.
Hum Mutat ; 40(7): 842-864, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30882951

RESUMO

Mutations in the GNPTAB and GNPTG genes cause mucolipidosis (ML) type II, type III alpha/beta, and type III gamma, which are autosomal recessively inherited lysosomal storage disorders. GNPTAB and GNPTG encode the α/ß-precursor and the γ-subunit of N-acetylglucosamine (GlcNAc)-1-phosphotransferase, respectively, the key enzyme for the generation of mannose 6-phosphate targeting signals on lysosomal enzymes. Defective GlcNAc-1-phosphotransferase results in missorting of lysosomal enzymes and accumulation of non-degradable macromolecules in lysosomes, strongly impairing cellular function. MLII-affected patients have coarse facial features, cessation of statural growth and neuromotor development, severe skeletal abnormalities, organomegaly, and cardiorespiratory insufficiency leading to death in early childhood. MLIII alpha/beta and MLIII gamma are attenuated forms of the disease. Since the identification of the GNPTAB and GNPTG genes, 564 individuals affected by MLII or MLIII have been described in the literature. In this report, we provide an overview on 258 and 50 mutations in GNPTAB and GNPTG, respectively, including 58 novel GNPTAB and seven novel GNPTG variants. Comprehensive functional studies of GNPTAB missense mutations did not only gain insights into the composition and function of the GlcNAc-1-phosphotransferase, but also helped to define genotype-phenotype correlations to predict the clinical outcome in patients.


Assuntos
Mucolipidoses/genética , Mutação , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Éxons , Humanos , Íntrons , Doenças por Armazenamento dos Lisossomos do Sistema Nervoso/classificação , Doenças por Armazenamento dos Lisossomos do Sistema Nervoso/genética , Mucolipidoses/classificação , Fenótipo , Prognóstico , Domínios Proteicos , Transferases (Outros Grupos de Fosfato Substituídos)/química
7.
Commun Biol ; 2: 36, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30701201

RESUMO

Colistin is the last-resort antibiotic against lethal infections with multidrug-resistant bacterial pathogens. A rainbow coalition of mobile colistin resistance (mcr) genes raises global health concerns. Here, we describe the action and mechanism of colistin resistance imparted by MCR-4, a recently-identified member from the broader MCR family. We found that MCR-4 originates from the silenced variant of Shewanella frigidimarina via progressive evolution and forms a phylogenetically-distinct group from the well-studied MCR-1/2 family. Domain-swapping experiments further confirmed that MCR-1 and MCR-4 transmembrane and catalytic domains are not functionally-interchangeable. However, structural and functional analyses demonstrated that MCR-4 possesses a similar PE lipid substrate-recognizable cavity and exploits an almost-identical ping-pong catalysis mechanism. MCR-4 also can alleviate colistin-triggered accumulation of reactive oxygen species (ROS). Taken together, this finding constitutes a functional proof that MCR-4 proceeds in a distinct evolutionary path to fulfill a consistent molecular mechanism, resulting in phenotypic colistin resistance.


Assuntos
Colistina/química , Transferases (Outros Grupos de Fosfato Substituídos)/química , Sequência de Aminoácidos , Colistina/metabolismo , Evolução Molecular , Mutação com Ganho de Função , Modelos Moleculares , Filogenia , Conformação Proteica , Proteólise , Relação Estrutura-Atividade , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo
8.
Int J Syst Evol Microbiol ; 69(3): 805-810, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30688631

RESUMO

A Gram-stain-negative, motile with single polar flagellum, rod-shaped bacterium, designated as strain DSL-35T, was isolated from the location where the ocean and Dishui lake meet at Shanghai on the East China Sea and characterized phylogenetically and phenotypically. Optimal growth occurred at 35 °C (range, 4-40 °C), pH 8 pH 5-11) and with 3-4 % (w/v) NaCl (0-12 %). Phylogenetic analysis based on its 16S rRNA gene sequence showed that strain DSL-35T was related to members of the genus Marinomonas and shared the highest sequence identities with Marinomonasarctica 328T (98.0 %), Marinomonashwangdonensis HDW-15T (97.5 %) and Marinomonasrhizomae IVIA-Po-145T (97.2 %). The 16S rRNA gene sequence identities between strain DSL-35T and other members of the genus Marinomonas were below 96.8 %. The digital DNA-DNA hybridization values between strain DSL-35T and the three type strains, Marinomonas. arctica 328T, M. rhizomae HDW-15T and M. rhizomae IVIA-Po-145T, were 30.9±2.4 %, 21.7±2.2% and 22±2.3 %, respectively. The average nucleotide identity values between strain DSL-35T and the three type strains were 87.6 %, 84.6 and 84.2 %, respectively. The predominant ubiquinone was Q-8. The major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The predominant cellular fatty acids of strain DSL-35T were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c; 40.0 %), C16 : 0 (22.5 %), summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c; 11.2 %), summed feature 2 (C14 : 0 3-OH and/or iso I C16 : 1; 7.2 %), C14 : 0 (6.8 %) and C12 : 0 (5.2 %). The G+C content of the genomic DNA was 44.5 mol%. The combined genotypic and phenotypic data indicated that strain DSL-35T represents a novel species of the genus Marinomonas, for which the name Marinomonas shanghaiensis sp. nov. is proposed, with the type strain DSL-35T (=KCTC 62646T=MCCC 1K03535T).


Assuntos
Lagos/microbiologia , Marinomonas/classificação , Filogenia , Água do Mar/microbiologia , Técnicas de Tipagem Bacteriana , Composição de Bases , China , DNA Bacteriano/genética , Ácidos Graxos/química , Marinomonas/isolamento & purificação , Hibridização de Ácido Nucleico , Oceanos e Mares , Fosfatidiletanolaminas/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Transferases (Outros Grupos de Fosfato Substituídos)/química , Ubiquinona/química
9.
Biotechnol J ; 14(4)2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30367549

RESUMO

The availability of nucleotide sugars is considered as bottleneck for Leloir-glycosyltransferases mediated glycan synthesis. A breakthrough for the synthesis of nucleotide sugars is the development of salvage pathway like enzyme cascades with high product yields from affordable monosaccharide substrates. In this regard, the authors aim at high enzyme productivities of these cascades by a repetitive batch approach. The authors report here for the first time that the exceptional high enzyme cascade stability facilitates the synthesis of Uridine-5'-diphospho-α-d-galactose (UDP-Gal), Uridine-5'-diphospho-N-acetylglucosamine (UDP-GlcNAc), and Uridine-5'-diphospho-N-acetylgalactosamine (UDP-GalNAc) in a multi-gram scale by repetitive batch mode. The authors obtained 12.8 g UDP-Gal through a high mass based total turnover number (TTNmass ) of 494 [gproduct /genzyme ] and space-time-yield (STY) of 10.7 [g/L*h]. Synthesis of UDP-GlcNAc in repetitive batch mode gave 11.9 g product with a TTNmass of 522 [gproduct /genzyme ] and a STY of 9.9 [g/L*h]. Furthermore, the scale-up to a 200 mL scale using a pressure operated concentrator was demonstrated for a UDP-GalNAc producing enzyme cascade resulting in an exceptional high STY of 19.4 [g/L*h] and 23.3 g product. In conclusion, the authors demonstrate that repetitive batch mode is a versatile strategy for the multi-gram scale synthesis of nucleotide sugars by stable enzyme cascades.


Assuntos
Polissacarídeos/química , Uridina Difosfato Galactose/biossíntese , Uridina Difosfato N-Acetilglicosamina/biossíntese , Açúcares de Uridina Difosfato/biossíntese , Glicosiltransferases/química , Nucleotídeos/biossíntese , Nucleotídeos/química , Transferases (Outros Grupos de Fosfato Substituídos)/química , Uridina Difosfato Galactose/química , Uridina Difosfato N-Acetilglicosamina/química , Açúcares de Uridina Difosfato/química
10.
BMC Vet Res ; 14(1): 416, 2018 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-30591066

RESUMO

BACKGROUND: Mucolipidosis II (ML II; I-cell disease) is caused by a deficiency of N-acetylglucosamine-1-phosphotransferase (GNPTAB; EC 2.7.8.17), which leads to a failure to internalize acid hydrolases into lysosomes for proper catabolism of various substances. This is an autosomal recessive lysosomal storage disease and causes severe progressive neuropathy and oculoskeletal dysfunction in humans (OMIM 252500). A naturally occurring disease model has been reported in juvenile domestic cats (OMIA 001248-9685) with clinical signs similar to human patients. We investigated the molecular genetic basis of ML II in a colony of affected cats by sequencing the coding and regulatory regions of GNPTAB from affected and clinically healthy related and unrelated domestic cats and compared the sequences to the published feline genome sequence (NCBI-RefSeq accession no. XM_003989173.4, Gene ID: 101100231). RESULTS: All affected cats were homozygous for a single base substitution (c.2644C > T) in exon 13 of GNPTAB. This variant results in a premature stop codon (p.Gln882*) which predicts severe truncation and complete dysfunction of the GNPTAB enzyme. About 140 GNPTAB variants have been described in human ML II patients, with 41.3% nonsense/missense mutations, nine occurring in the same gene region as in this feline model. Restriction fragment length polymorphism and allelic discrimination real-time polymerase chain reaction assays accurately differentiated between clear, asymptomatic carriers and homozygous affected cats. CONCLUSION: Molecular genetic characterization advances this large animal model of ML II for use to further define the pathophysiology of the disease and evaluate novel therapeutic approaches for this fatal lysosomal storage disease in humans.


Assuntos
Doenças do Gato/enzimologia , Doenças do Gato/genética , Variação Genética , Mucolipidoses/veterinária , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Animais , Gatos , Códon de Terminação/genética , Modelos Animais de Doenças , Mucolipidoses/genética , Mutação , Transferases (Outros Grupos de Fosfato Substituídos)/química
11.
J Biol Chem ; 293(47): 18328-18336, 2018 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-30305392

RESUMO

2-Hydroxy-oleic acid (2OHOA) is a potent anticancer drug that induces cancer cell cycle arrest and apoptosis. Previous studies have suggested that 2OHOA's anticancer effect is mediated by SMS activation in cancer cells, including A549 and U118 cells. To confirm this phenomenon, in this study, we treated both A549 and U118 cells with 2OHOA and measured SMS activity. To our surprise, we found neither 2OHOA-mediated SMS activation nor sphingomyelin accumulation in the cells. However, we noted that 2OHOA significantly reduces phosphatidylcholine in these cells. We also did not observe 2OHOA-mediated SMS activation in mouse tissue homogenates. Importantly, 2OHOA inhibited rather than activated recombinant SMS1 (rSMS1) and rSMS2 in a dose-dependent fashion. Intra-gastric treatment of C57BL/6J mice with 2OHOA for 10 days had no effects on liver and small intestine SMS activities and plasma sphingomyelin levels. The treatment inhibited lysophosphatidylcholine acyltransferase (LPCAT) activity, consistent with the aforementioned reduction in plasma phosphatidylcholine. Because total cellular phosphatidylcholine is used as a predictive biomarker for monitoring tumor responses, the previously reported 2OHOA-mediated cancer suppression could be related to this phosphatidylcholine reduction, which may influence cell membrane structure and properties. We conclude that 2OHOA is not a SMS activator and that its anticancer property may be related to an effect on phosphatidylcholine metabolism.


Assuntos
Antineoplásicos/metabolismo , Neoplasias/enzimologia , Ácidos Oleicos/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Linhagem Celular Tumoral , Ativação Enzimática , Ativadores de Enzimas/administração & dosagem , Ativadores de Enzimas/química , Ativadores de Enzimas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Ácidos Oleicos/administração & dosagem , Ácidos Oleicos/química , Fosfatidilcolinas/metabolismo , Esfingomielinas/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/genética
12.
Angew Chem Int Ed Engl ; 57(39): 12666-12669, 2018 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-30080959

RESUMO

Covalent surface immobilization of proteins for binding assays is typically performed non-specifically via lysine residues. However, receptors that either have lysines near their binding pockets, or whose presence at the sensor surface is electrostatically disfavoured, can be hard to probe. To overcome these limitations and to improve the homogeneity of surface functionalization, we adapted and optimized three different enzymatic coupling strategies (4'-phosphopantetheinyl transferase, sortase A, and asparaginyl endopeptidase) for biolayer interferometry surface modification. All of these enzymes can be used to site-specifically and covalently ligate proteins of interest via short recognition sequences. The enzymes function under mild conditions and thus immobilization does not affect the receptors' functionality. We successfully employed this enzymatic surface functionalization approach to study the binding kinetics of two different receptor-ligand pairs.


Assuntos
Aminoaciltransferases/química , Proteínas de Bactérias/química , Cisteína Endopeptidases/química , Transferases (Outros Grupos de Fosfato Substituídos)/química , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Cinética , Ligação Proteica , Propriedades de Superfície , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo
13.
Drug Discov Today ; 23(7): 1426-1435, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29778697

RESUMO

The rapid growth of antibiotic-resistant bacterial infections is of major concern for human health. Therefore, it is of great importance to characterize novel targets for the development of antibacterial drugs. One promising protein target is MraY (UDP-N-acetylmuramyl-pentapeptide: undecaprenyl phosphate N-acetylmuramyl-pentapeptide-1-phosphate transferase or MurNAc-1-P-transferase), which is essential for bacterial cell wall synthesis. Here, we summarize recent breakthroughs in structural studies of bacterial MraYs and the closely related human GPT (UDP-N-acetylglucosamine: dolichyl phosphate N-acetylglucosamine-1-phosphate transferase or GlcNAc-1-P-transferase). We present a detailed comparison of interaction modes with the natural product inhibitors tunicamycin and muraymycin D2. Finally, we speculate on possible routes to design an antibacterial agent in the form of a potent and selective inhibitor against MraY.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Proteínas de Bactérias/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Peptidoglicano/biossíntese , Transferases/antagonistas & inibidores , Animais , Antibacterianos/síntese química , Bactérias/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana , Inibidores Enzimáticos/síntese química , Humanos , Modelos Moleculares , Nucleosídeos/química , Nucleosídeos/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Conformação Proteica , Relação Estrutura-Atividade , Transferases/química , Transferases/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Tunicamicina/química , Tunicamicina/farmacologia
14.
ACS Chem Biol ; 13(5): 1322-1332, 2018 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-29631403

RESUMO

Lipid A phosphoethanolamine (PEtN) transferases render bacteria resistant to the last resort antibiotic colistin. The recent discoveries of pathogenic bacteria harboring plasmid-borne PEtN transferase ( mcr) genes have illustrated the serious potential for wide dissemination of these resistance elements. The origin of mcr-1 is traced to Moraxella species co-occupying environmental niches with Enterobacteriaceae. Here, we describe the crystal structure of the catalytic domain of the chromosomally encoded colistin resistance PEtN transferase, ICR Mc (for intrinsic colistin resistance) of Moraxella catarrhalis. The ICR Mc structure in complex with PEtN reveals key molecular details including specific residues involved in catalysis and PEtN binding. It also demonstrates that ICR Mc catalytic domain dimerization is required for substrate binding. Our structure-guided phylogenetic analysis provides sequence signatures defining potentially colistin-active representatives in this enzyme family. Combined, these results advance the molecular and mechanistic understanding of PEtN transferases and illuminate their origins.


Assuntos
Colistina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Moraxella catarrhalis/enzimologia , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Antibacterianos/farmacologia , Cristalografia por Raios X , Dimerização , Lipídeo A/metabolismo , Polimixinas/farmacologia , Conformação Proteica , Especificidade por Substrato , Transferases (Outros Grupos de Fosfato Substituídos)/química
15.
ACS Synth Biol ; 7(4): 1143-1151, 2018 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-29562128

RESUMO

Phosphopantetheinyl transferases catalyze the post-translational modification of carrier proteins involved in both primary and secondary metabolism. The functional expression of polyketide synthases and nonribosomal peptide synthetases requires the activation of all carrier protein domains by phosphopantetheinyl transferases. Here we describe the characterization of five bacterial phosphopantetheinyl transferases by their substrate specificity and catalytic efficiency of four cyanobacterial carrier proteins. Comparative in vitro phosphopantetheinylation analysis showed Sfp possesses the highest catalytic efficiency over various carrier proteins. In vivo coexpression of phosphopantetheinyl transferases with carrier proteins revealed a broad range substrate specificity of phosphopantetheinyl transferases; all studied phosphopantetheinyl transferases were capable of converting apo- carrier proteins, sourced from diverse biosynthetic enzymes, to their active holo form. Phosphopantetheinyl transferase coexpression with the hybrid nonribosomal peptide synthetases/polyketide synthases responsible for microcystin biosynthesis confirmed that the higher in vitro activity of Sfp translated in vivo to a higher yield of production.


Assuntos
Proteínas de Bactérias/metabolismo , Produtos Biológicos/metabolismo , Proteínas de Transporte/metabolismo , Cianobactérias/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Simulação por Computador , Cianobactérias/química , Ligação de Hidrogênio , Microcistinas/genética , Microcistinas/metabolismo , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Policetídeo Sintases/metabolismo , Processamento de Proteína Pós-Traducional , Especificidade por Substrato , Transferases (Outros Grupos de Fosfato Substituídos)/genética
16.
Nat Struct Mol Biol ; 25(3): 217-224, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29459785

RESUMO

N-linked glycosylation is a predominant post-translational modification of protein in eukaryotes, and its dysregulation is the etiology of several human disorders. The enzyme UDP-N-acetylglucosamine:dolichyl-phosphate N-acetylglucosaminephosphotransferase (GlcNAc-1-P-transferase or GPT) catalyzes the first and committed step of N-linked glycosylation in the endoplasmic reticulum membrane, and it is the target of the natural product tunicamycin. Tunicamycin has potent antibacterial activity, inhibiting the bacterial cell wall synthesis enzyme MraY, but its usefulness as an antibiotic is limited by off-target inhibition of human GPT. Our understanding of how tunicamycin inhibits N-linked glycosylation and efforts to selectively target MraY are hampered by a lack of structural information. Here we present crystal structures of human GPT in complex with tunicamycin. Structural and functional analyses reveal the difference between GPT and MraY in their mechanisms of inhibition by tunicamycin. We demonstrate that this difference could be exploited to design MraY-specific inhibitors as potential antibiotics.


Assuntos
Antibacterianos/química , Inibidores Enzimáticos/química , Transferases (Outros Grupos de Fosfato Substituídos)/química , Tunicamicina/química , Antibacterianos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Inibidores Enzimáticos/metabolismo , Glicosilação , Humanos , Magnésio/química , Modelos Moleculares , Ligação Proteica , Multimerização Proteica , Especificidade por Substrato , Transferases/química , Transferases/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Tunicamicina/metabolismo
17.
Cell Tissue Res ; 372(1): 33-40, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29294205

RESUMO

Sphingomyelin synthase genes (Sgms1 and Sgms2) encode the vital enzymes that participate in the processes of membrane transport, cell proliferation and apoptosis. We previously determined the exon-intron structure of Sgms1 and some features of its expression in human and rodent tissues. The circular RNAs (circRNAs) emerging from exons of the 5'-untranslated region (5'-UTR) of Sgms1 were determined. These circRNAs are represented at a high level in the adult brain. Here, we demonstrate that, in contrast to Sgms1, Sgms2 does not contain the multi-exon 5'-UTR but encodes circRNAs, which are composed of the coding region of the gene and are expressed at a low level. We present a study of the expression of sphingomyelin synthase genes in rat brain at embryonic days 7, 9, 13, 17 and 21 and in adult rat brain. In contrast to Sgms1, Sgms2 is expressed at a significantly low level in adult brain. In embryonic rat brain, the mRNA expression of sphingomyelin synthase genes is varied in a developmental stage-specific manner. The level of Sgms1 mRNAs, differing by 5'-UTR-in the formation of which alternative promoters can participate-changes significantly during the process of embryonic development. The expression of circRNAs of Sgms1 was significantly raised during rat embryonic brain development. We assume that the circRNAs are involved in the regulation of sphingomyelin synthase activity in rat brain in different developmental stages.


Assuntos
Encéfalo/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Regiões 5' não Traduzidas/genética , Animais , Biologia Computacional , Embrião de Mamíferos/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Regiões Promotoras Genéticas , RNA , RNA Circular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Wistar , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo
18.
Int J Syst Evol Microbiol ; 68(1): 125-130, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29116033

RESUMO

A Gram-stain-negative, aerobic, non-motile, non-spore-forming and rod-shaped bacterial strain, designated HKS-05T, was isolated from ginseng field soil. This bacterium was characterized to determine its taxonomic position by using the polyphasic approach. HKS-05T grew at 10-37 °C and at pH 6.0-8.0 on R2A agar. On the basis of 16S rRNA gene sequence similarity, HKS-05T was shown to represent a member of the family Caulobacteraceaeand to be related to Phenylobacterium lituiforme FaiI3T (98.1 % sequence similarity), 'Phenylobacterium zucineum' HLK1 (97.9 %), Phenylobacterium muchangponense A8T (97.7 %), Phenylobacteriumcomposti 4T-6T (97.2 %) and Phenylobacterium immobile ET (97.1 %). The major respiratory quinone was Q-10 and the major fatty acids were summed feature 8 (comprising C18 : 1ω7c and/or C18 : 1ω6c), C16 : 0, and summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c). The polar lipids were phosphatidylglycerol, unidentified glycolipids and unidentified polar lipids. The G+C content of the genomic DNA was 70.4 mol%. DNA-DNA relatedness values between HKS-05T and its closest phylogenetically neighbours were low. HKS-05T could be differentiated genotypically and phenotypically from the species of the genus Phenylobacterium with validly published names. The isolate therefore represents a novel species, for which the name Phenylobacteriumhankyongense sp. nov. is proposed, with the type strain HKS-05T (=KACC 18628T=LMG 30081T).


Assuntos
Caulobacteraceae/classificação , Panax/microbiologia , Filogenia , Microbiologia do Solo , Técnicas de Tipagem Bacteriana , Composição de Bases , Caulobacteraceae/genética , Caulobacteraceae/isolamento & purificação , DNA Bacteriano/genética , Ácidos Graxos/química , Glicolipídeos/química , Hibridização de Ácido Nucleico , RNA Ribossômico 16S/genética , República da Coreia , Análise de Sequência de DNA , Transferases (Outros Grupos de Fosfato Substituídos)/química , Ubiquinona/química
19.
Glycobiology ; 28(2): 100-107, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29228283

RESUMO

Neisseria meningitidis Group X is an emerging cause of bacterial meningitis in Sub-Saharan Africa. The capsular polysaccharide of Group X is a homopolymer of N-acetylglucosamine α(1-4) phosphate and is a vaccine target for prevention of disease associated with this meningococcal serogroup. We have demonstrated previously that the formation of the polymer is catalyzed by a phosphotransferase which transfers N-acetylglucosamine-1-phosphate from UDP-N-acetylglucosamine to the 4-hydroxyl of the N-acetylglucosamine on the nonreducing end of the growing chain. In this study, we use substrate analogs of UDP-GlcNAc to define the enzyme/donor substrate interactions critical for catalysis. Our kinetic analysis of the phosphotransferase reaction is consistent with a sequential mechanism of substrate addition and product release. The use of novel uracil modified analogs designed by Wagner et al. enabled us to assess whether the CsxA-catalyzed reaction is consistent with a donor dependent conformational change. As expected with this model for glycosyltransferases, UDP-GlcNAc analogs with bulky uracil modifications are not substrates but are inhibitors. An analog with a smaller iodo uracil substitution is a substrate and a less potent inhibitor. Moreover, our survey of analogs with modifications on the N-acetylglucosamine residue of the sugar nucleotide donor highlights the importance of substituents at C2 and C4 of the sugar residue. The hydroxyl group at C4 and the structure of the acyl group at C2 are very important for specificity and substrate interactions during the polymerization reaction. While most analogs modified at C2 were inhibitors, acetamido analogs were also substrates suggesting the importance of the carbonyl group.


Assuntos
Proteínas de Bactérias/metabolismo , Neisseria meningitidis/enzimologia , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Cápsulas Bacterianas/metabolismo , Proteínas de Bactérias/química , Polissacarídeos Bacterianos/metabolismo , Ligação Proteica , Transferases (Outros Grupos de Fosfato Substituídos)/química
20.
Mol Microbiol ; 107(5): 623-638, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29280215

RESUMO

Osmosensing by transporter ProP is modulated by its cardiolipin (CL)-dependent concentration at the poles of Escherichia coli cells. Other contributors to this phenomenon were sought with the BACterial Two-Hybrid System (BACTH). The BACTH-tagged variants T18-ProP and T25-ProP retained ProP function and localization. Their interaction confirmed the ProP homo-dimerization previously established by protein crosslinking. YdhP, YjbJ and ClsA were prominent among the putative ProP interactors identified by the BACTH system. The functions of YdhP and YjbJ are unknown, although YjbJ is an abundant, osmotically induced, soluble protein. ClsA (CL Synthase A) had been shown to determine ProP localization by mediating CL synthesis. Unlike a deletion of clsA, deletion of ydhP or yjbJ had no effect on ProP localization or function. All three proteins were concentrated at the cell poles, but only ClsA localization was CL-dependent. ClsA was shown to be N-terminally processed and membrane-anchored, with dual, cytoplasmic, catalytic domains. Active site amino acid replacements (H224A plus H404A) inactivated ClsA and compromised ProP localization. YdhP and YjbJ may be ClsA effectors, and interactions of YdhP, YjbJ and ClsA with ProP may reflect their colocalization at the cell poles. Targeted CL synthesis may contribute to the polar localization of CL, ClsA and ProP.


Assuntos
Cardiolipinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Proteínas de Membrana/metabolismo , Osmorregulação , Simportadores/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Citoplasma/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Deleção de Genes , Proteínas de Membrana/química , Proteínas de Membrana/genética , Concentração Osmolar , Conformação Proteica , Multimerização Proteica , Simportadores/química , Simportadores/genética , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/genética
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