RESUMEN
Processing of RNA is a key regulatory mechanism for all living systems. Escherichia coli protein YicC belongs to the well-conserved YicC family and has been identified as a novel ribonuclease. Here, we report a 2.8-Å-resolution crystal structure of the E. coli YicC apo protein and a 3.2-Å-cryo-EM structure of YicC bound to an RNA substrate. The apo YicC forms a dimer of trimers with a large open channel. In the RNA-bound form, the top trimer of YicC rotates nearly 70° and closes the RNA substrate inside the cavity to form a clamshell-pearl conformation that resembles no other known RNases. The structural information combined with mass spectrometry and biochemical data identified cleavage on the upstream side of an RNA hairpin. Mutagenesis studies demonstrated that the previously uncharacterized domain, DUF1732, is critical in both RNA binding and catalysis. These studies shed light on the mechanism of the previously unexplored YicC RNase family.
RESUMEN
Glutaric Aciduria Type 1 (GA1) is a serious inborn error of metabolism with no pharmacological treatments. A novel strategy to treat this disease is to divert the toxic biochemical intermediates to less toxic or nontoxic metabolites. Here, we report a putative novel target, succinyl-CoA:glutarate-CoA transferase (SUGCT), which we hypothesize suppresses the GA1 metabolic phenotype through decreasing glutaryl-CoA and the derived 3-hydroxyglutaric acid. SUGCT is a type III CoA transferase that uses succinyl-CoA and glutaric acid as substrates. We report the structure of SUGCT, develop enzyme- and cell-based assays, and identify valsartan and losartan carboxylic acid as inhibitors of the enzyme in a high-throughput screen of FDA-approved compounds. The cocrystal structure of SUGCT with losartan carboxylic acid revealed a novel pocket in the active site and further validated the high-throughput screening approach. These results may form the basis for the future development of new pharmacological intervention to treat GA1.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos , Encefalopatías Metabólicas , Humanos , Errores Innatos del Metabolismo de los Aminoácidos/tratamiento farmacológico , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Errores Innatos del Metabolismo de los Aminoácidos/enzimología , Errores Innatos del Metabolismo de los Aminoácidos/genética , Encefalopatías Metabólicas/tratamiento farmacológico , Encefalopatías Metabólicas/metabolismo , Encefalopatías Metabólicas/enzimología , Glutaratos/metabolismo , Glutaratos/química , Losartán/farmacología , Losartán/química , Coenzima A Transferasas/metabolismo , Coenzima A Transferasas/antagonistas & inhibidores , Coenzima A Transferasas/genética , Coenzima A Transferasas/química , Valsartán , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Cristalografía por Rayos X , Dominio Catalítico , Acilcoenzima A/metabolismo , Acilcoenzima A/química , Modelos Moleculares , Ensayos Analíticos de Alto Rendimiento , Glutaril-CoA Deshidrogenasa/deficienciaRESUMEN
Glutaric Aciduria Type 1 (GA1) is a serious inborn error of metabolism with no pharmacological treatments. A novel strategy to treat this disease is to divert the toxic biochemical intermediates to less toxic or non-toxic metabolites. Here, we report a novel target, SUGCT, which we hypothesize suppresses the GA1 metabolic phenotype through decreasing glutaryl-CoA. We report the structure of SUGCT, the first eukaryotic structure of a type III CoA transferase, develop a high-throughput enzyme assay and a cell-based assay, and identify valsartan and losartan carboxylic acid as inhibitors of the enzyme validating the screening approach. These results may form the basis for future development of new pharmacological intervention to treat GA1.
RESUMEN
Hyperlysinemia is a rare autosomal recessive deficiency of 2-aminoadipic semialdehyde synthase (AASS) affecting the initial step in lysine degradation. It is thought to be a benign biochemical abnormality, but reports on cases remain scarce. The description of additional cases, in particular, those identified without ascertainment bias, may help counseling of new cases in the future. It may also help to establish the risks associated with pharmacological inhibition of AASS, a potential therapeutic strategy that is under investigation for other inborn errors of lysine degradation. We describe the identification of a hyperlysinemia case identified in the Provincial Neonatal Urine Screening Program in Sherbrooke, Quebec. This case presented with a profile of cystinuria but with a very high increase in urinary lysine. A diagnosis of hyperlysinemia was confirmed through biochemical testing and the identification of biallelic variants in AASS. The p.R146W and p.T371I variants are novel and affect the folding of the lysine-2-oxoglutarate domain of AASS. The 11-month-old boy is currently doing well without any therapeutic interventions. The identification of this case through newborn urine screening further establishes that hyperlysinemia is a biochemical abnormality with limited clinical consequences and may not require any intervention.
RESUMEN
Autophagy is a conserved metabolic pathway that is central to many diseases. Recently, there has been a lot of interest in targeting autophagy with small molecule inhibitors as a possible therapeutic strategy. However, many of the compounds used for autophagy are nonselective. Here, we explored the inhibition of autophagy in pancreatic cancer cells using established selective small molecule inhibitors and discovered an unexpected link between the autophagy pathway and progression through the cell cycle. Our findings revealed that treatments with inhibitors that have different autophagy pathway targets block cell replication and activate other metabolic pathways to compensate for the blockade in autophagy. An unbiased screen looking for known drugs that might synergize with autophagy inhibition revealed new combination treatments that might provide a blueprint for therapeutic approaches to pancreatic cancer. The drugs quizartinib and THZ1 showed a strong synergistic effect in pancreatic cells with autophagy inhibition.
Asunto(s)
Autofagia , Ciclo Celular , Neoplasias Pancreáticas , Humanos , Autofagia/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Combinación de Medicamentos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias PancreáticasRESUMEN
In humans, a single enzyme 2-aminoadipic semialdehyde synthase (AASS) catalyses the initial two critical reactions in the lysine degradation pathway. This enzyme evolved to be a bifunctional enzyme with both lysine-2-oxoglutarate reductase (LOR) and saccharopine dehydrogenase domains (SDH). Moreover, AASS is a unique drug target for inborn errors of metabolism such as glutaric aciduria type 1 that arise from deficiencies downstream in the lysine degradation pathway. While work has been done to elucidate the SDH domain structurally and to develop inhibitors, neither has been done for the LOR domain. Here, we purify and characterize LOR and show that it is activated by alkylation of cysteine 414 by N-ethylmaleimide. We also provide evidence that AASS is rate-limiting upon high lysine exposure of mice. Finally, we present the crystal structure of the human LOR domain. Our combined work should enable future efforts to identify inhibitors of this novel drug target.
Asunto(s)
Lisina , Sacaropina Deshidrogenasas , Errores Innatos del Metabolismo de los Aminoácidos , Animales , Encefalopatías Metabólicas , Cisteína , Etilmaleimida , Glutaril-CoA Deshidrogenasa/deficiencia , Humanos , Lisina/metabolismo , Ratones , Sacaropina Deshidrogenasas/química , Sacaropina Deshidrogenasas/metabolismoRESUMEN
The Bacillus subtilis genome is predicted to encode numerous ribonucleases, including four 3' exoribonucleases that have been characterized to some extent. A strain containing gene knockouts of all four known 3' exoribonucleases is viable, suggesting that one or more additional RNases remain to be discovered. A protein extract from the quadruple RNase mutant strain was fractionated and RNase activity was followed, resulting in the identification of an enzyme activity catalyzed by the YloC protein. YloC is an endoribonuclease and is a member of the highly conserved "YicC family" of proteins that is widespread in bacteria. YloC is a metal-dependent enzyme that catalyzes the cleavage of single-stranded RNA, preferentially at U residues, and exists in an oligomeric form, most likely a hexamer. As such, YloC shares some characteristics with the SARS-CoV Nsp15 endoribonuclease. While the in vivo function of YloC in B. subtilis is yet to be determined, YloC was found to act similarly to YicC in an Escherichia coli in vivo assay that assesses decay of the small RNA, RyhB. Thus, YloC may play a role in small RNA regulation.
Asunto(s)
Bacillus subtilis/genética , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Bacillus subtilis/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Endorribonucleasas/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Microorganismos Modificados Genéticamente , Mutación , Estabilidad del ARN , ARN Bacteriano/química , ARN Bacteriano/metabolismo , Ribonucleasas/genética , Ribonucleasas/metabolismo , Especificidad por Sustrato , Proteínas no Estructurales Virales/metabolismoRESUMEN
Antibiotics to treat drug-resistant Gram-negative infections are urgently needed but challenging to discover. Using a cell-based screen, we identified a simple secondary amine that inhibited the growth of wild-type Escherichia coli and Acinetobacter baumannii but not the growth of the Gram-positive organism Bacillus subtilis. Resistance mutations in E. coli and A. baumannii mapped exclusively to the aminoacyl-tRNA synthetase PheRS. We confirmed biochemically that the compound inhibited PheRS from these organisms and showed that it did not inhibit PheRS from B. subtilis or humans. To understand the basis for the compound's high selectivity for only some PheRS enzymes, we solved crystal structures of E. coli and A. baumannii PheRS complexed with the inhibitor. The structures showed that the compound's benzyl group mimics the benzyl of phenylalanine. The other amine substituent, a 2-(cyclohexen-1-yl)ethyl group, induces a hydrophobic pocket in which it binds. Through bioinformatic analysis and mutagenesis, we show that the ability to induce a complementary hydrophobic pocket that can accommodate the second substituent explains the high selectivity of this remarkably simple molecular scaffold for Gram-negative PheRS. Because this secondary amine scaffold is active against wild-type Gram-negative pathogens but is not cytotoxic to mammalian cells, we suggest that it may be possible to develop it for use in combination antibiotic therapy to treat Gram-negative infections.
Asunto(s)
Aminas/farmacología , Antibacterianos/farmacología , Bacillus subtilis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Fenilalanina-ARNt Ligasa/antagonistas & inhibidores , Aminas/química , Antibacterianos/química , Bacillus subtilis/enzimología , Inhibidores Enzimáticos/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Fenilalanina-ARNt Ligasa/metabolismoRESUMEN
DHTKD1 is the E1 component of the 2-oxoadipate dehydrogenase complex, which is an enzyme involved in the catabolism of (hydroxy-)lysine and tryptophan. Mutations in DHTKD1 have been associated with 2-aminoadipic and 2-oxoadipic aciduria, Charcot-Marie-Tooth disease type 2Q and eosinophilic esophagitis, but the pathophysiology of these clinically distinct disorders remains elusive. Here, we report the identification of adipoylphosphonic acid and tenatoprazole as DHTKD1 inhibitors using targeted and high throughput screening, respectively. We furthermore elucidate the DHTKD1 crystal structure with thiamin diphosphate bound at 2.25 Å. We also report the impact of 10 disease-associated missense mutations on DHTKD1. Whereas the majority of the DHTKD1 variants displayed impaired folding or reduced thermal stability in combination with absent or reduced enzyme activity, three variants showed no abnormalities. Our work provides chemical and structural tools for further understanding of the function of DHTKD1 and its role in several human pathologies.
Asunto(s)
Complejo Cetoglutarato Deshidrogenasa/antagonistas & inhibidores , Tiamina Pirofosfato/química , Dicroismo Circular , Cristalografía por Rayos X , Humanos , Complejo Cetoglutarato Deshidrogenasa/química , Complejo Cetoglutarato Deshidrogenasa/genética , Estructura Molecular , Mutación MissenseRESUMEN
The ULK (UNC51-like) enzymes are a family of mammalian kinases that have critical roles in autophagy and development. While ULK1, ULK2, and ULK3 have been characterized, very little is known about ULK4. However, recently, deletions in ULK4 have been genetically linked to increased susceptibility to developing schizophrenia, a devastating neuropsychiatric disease with high heritability but few genes identified. Interestingly, ULK4 is a pseudokinase with some unusual mutations in the kinase catalytic motifs. Here, we report the first structure of the human ULK4 kinase at high resolution and show that although ULK4 has no apparent phosphotransfer activity, it can strongly bind ATP. We find an unusual mechanism for binding ATP in a Mg2+-independent manner, including a rare hydrophobic bridge in the active site. In addition, we develop two assays for ATP binding to ULK4, perform a virtual and experimental screen to identify small-molecule binders of ULK4, and identify several novel scaffolds that bind ULK4 and can lead the way to more selective small molecules that may help shed light on the function of this enigmatic protein.
Asunto(s)
Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/química , Esquizofrenia/enzimología , Adenosina Trifosfato/metabolismo , Animales , Autofagia , Inhibidores Enzimáticos/farmacología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Mutación , Conformación Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Reversible glycosylation of nuclear and cytoplasmic proteins is an important regulatory mechanism across metazoans. One enzyme, O-linked N-acetylglucosamine transferase (OGT), is responsible for all nucleocytoplasmic glycosylation and there is a well-known need for potent, cell-permeable inhibitors to interrogate OGT function. Here we report the structure-based evolution of OGT inhibitors culminating in compounds with low nanomolar inhibitory potency and on-target cellular activity. In addition to disclosing useful OGT inhibitors, the structures we report provide insight into how to inhibit glycosyltransferases, a family of enzymes that has been notoriously refractory to inhibitor development.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , Células HCT116 , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , N-Acetilglucosaminiltransferasas/química , N-Acetilglucosaminiltransferasas/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Novobiocin is an orally active antibiotic that inhibits DNA gyrase by binding the ATP-binding site in the ATPase subunit. Although effective against Gram-positive pathogens, novobiocin has limited activity against Gram-negative organisms due to the presence of the lipopolysaccharide-containing outer membrane, which acts as a permeability barrier. Using a novobiocin-sensitive Escherichia coli strain with a leaky outer membrane, we identified a mutant with increased resistance to novobiocin. Unexpectedly, the mutation that increases novobiocin resistance was not found to alter gyrase, but the ATPase that powers lipopolysaccharide (LPS) transport. Co-crystal structures, biochemical, and genetic evidence show novobiocin directly binds this ATPase. Novobiocin does not bind the ATP binding site but rather the interface between the ATPase subunits and the transmembrane subunits of the LPS transporter. This interaction increases the activity of the LPS transporter, which in turn alters the permeability of the outer membrane. We propose that novobiocin will be a useful tool for understanding how ATP hydrolysis is coupled to LPS transport.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Antibacterianos/metabolismo , Lipopolisacáridos/metabolismo , Novobiocina/metabolismo , Novobiocina/farmacología , Adenosina Trifosfato/metabolismo , Sitios de Unión , Transporte Biológico/efectos de los fármacos , Girasa de ADN/metabolismo , Activación Enzimática/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Escherichia coli/genética , Hidrólisis/efectos de los fármacosRESUMEN
Elongation Factor-2 Kinase (eEF2K) in an unusual mammalian enzyme that has one known substrate, elongation factor-2. It belongs to a class of kinases, called alpha kinases, that has little sequence identity to the >500 conventional protein kinases, but performs the same reaction and has similar catalytic residues. The phosphorylation of eEF2 blocks translation elongation, which is thought to be critical to regulating cellular energy usage. Here we report a system for discovering new substrates of alpha kinases and identify the first new substrates of eEF2K including AMPK and alpha4, and determine a sequence motif for the kinase that shows a requirement for threonine residues as the target of phosphorylation. These new substrates suggest that eEF2K has a more diverse role in regulating cellular energy usage that involves multiple pathways and regulatory feedback.
Asunto(s)
Células/metabolismo , Quinasa del Factor 2 de Elongación/metabolismo , Secuencia de Aminoácidos , Biología Computacional , Quinasa del Factor 2 de Elongación/química , Células HeLa , Humanos , Péptidos/química , Péptidos/metabolismo , Fosforilación , Reproducibilidad de los Resultados , Especificidad por SustratoRESUMEN
The essential human enzyme O-linked ß-N-acetylglucosamine transferase (OGT), known for modulating the functions of nuclear and cytoplasmic proteins through serine and threonine glycosylation, was unexpectedly implicated in the proteolytic maturation of the cell cycle regulator host cell factor-1 (HCF-1). Here we show that HCF-1 cleavage occurs via glycosylation of a glutamate side chain followed by on-enzyme formation of an internal pyroglutamate, which undergoes spontaneous backbone hydrolysis.
Asunto(s)
Amidas/química , Amidas/metabolismo , Biocatálisis , Factor C1 de la Célula Huésped/química , Factor C1 de la Célula Huésped/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Humanos , HidrólisisRESUMEN
Energy homeostasis in eukaryotic cells is a complex and fundamental process that is misregulated in several human diseases. A key component of energy regulation is a process called autophagy that involves the recycling of cellular components. There has been much recent interest in studying the mechanism of autophagy to understand an important cellular process and to evaluate the therapeutic potential in targeting autophagy. Activation of a kinase called ULK1 initiates autophagy by driving downstream pathways that lead to the formation of double membrane bound vesicles that surround the cellular contents that are to be degraded. Here, we report the discovery of an inhibitor of ULK1 with improved selectivity and a high-resolution crystal structure of the compound bound to the kinase, which will be useful tools for studying autophagy in cells.
Asunto(s)
Autofagia/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia , Humanos , Estructura MolecularRESUMEN
O-GlcNAc transferase (OGT) is an essential mammalian enzyme that regulates numerous cellular processes through the attachment of O-linked N-acetylglucosamine (O-GlcNAc) residues to nuclear and cytoplasmic proteins. Its targets include kinases, phosphatases, transcription factors, histones, and many other intracellular proteins. The biology of O-GlcNAc modification is still not well understood, and cell-permeable inhibitors of OGT are needed both as research tools and for validating OGT as a therapeutic target. Here, we report a small molecule OGT inhibitor, OSMI-1, developed from a high-throughput screening hit. It is cell-permeable and inhibits protein O-GlcNAcylation in several mammalian cell lines without qualitatively altering cell surface N- or O-linked glycans. The development of this molecule validates high-throughput screening approaches for the discovery of glycosyltransferase inhibitors, and further optimization of this scaffold may lead to yet more potent OGT inhibitors useful for studying OGT in animal models.
Asunto(s)
Inhibidores Enzimáticos/farmacología , N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Células CHO , Permeabilidad de la Membrana Celular , Cricetulus , Inhibidores Enzimáticos/síntesis química , Ensayos Analíticos de Alto Rendimiento , Humanos , Concentración 50 Inhibidora , Lectinas/química , Lectinas/metabolismo , N-Acetilglucosaminiltransferasas/química , N-Acetilglucosaminiltransferasas/metabolismo , Bibliotecas de Moléculas Pequeñas/síntesis química , Uridina Difosfato/química , Uridina Difosfato/metabolismo , Uridina Difosfato N-Acetilglucosamina/química , Uridina Difosfato N-Acetilglucosamina/metabolismoRESUMEN
Autophagy is a conserved cellular process that involves the degradation of cellular components for energy maintenance and cytoplasmic quality control that has recently gained interest as a novel target for a variety of human diseases, including cancer. A prime candidate to determine the potential therapeutic benefit of targeting autophagy is the kinase ULK1, whose activation initiates autophagy. Here, we report the first structures of ULK1, in complex with multiple potent inhibitors. These structures show features unique to the enzyme and will provide a path for the rational design of selective compounds as cellular probes and potential therapeutics.
Asunto(s)
Autofagia/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Autofagia/fisiología , Homólogo de la Proteína 1 Relacionada con la Autofagia , Escherichia coli/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Modelos Moleculares , Unión Proteica , Conformación Proteica , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
The cell surface of Gram-negative bacteria contains lipopolysaccharides (LPS), which provide a barrier against the entry of many antibiotics. LPS assembly involves a multiprotein LPS transport (Lpt) complex that spans from the cytoplasm to the outer membrane. In this complex, an unusual ATP-binding cassette transporter is thought to power the extraction of LPS from the outer leaflet of the cytoplasmic membrane and its transport across the cell envelope. We introduce changes into the nucleotide-binding domain, LptB, that inactivate transporter function in vivo. We characterize these residues using biochemical experiments combined with high-resolution crystal structures of LptB pre- and post-ATP hydrolysis and suggest a role for an active site residue in phosphate exit. We also identify a conserved residue that is not required for ATPase activity but is essential for interaction with the transmembrane components. Our studies establish the essentiality of ATP hydrolysis by LptB to power LPS transport in cells and suggest strategies to inhibit transporter function away from the LptB active site.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfatasas/metabolismo , Biocatálisis , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Lipopolisacáridos/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfato/metabolismo , Aminoácidos/metabolismo , Transporte Biológico , Dominio Catalítico , Membrana Celular/metabolismo , Cristalografía por Rayos X , Proteínas de Escherichia coli/química , Hidrólisis , Viabilidad Microbiana , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Unión Proteica , Estructura Secundaria de ProteínaRESUMEN
Host cell factor-1 (HCF-1), a transcriptional co-regulator of human cell-cycle progression, undergoes proteolytic maturation in which any of six repeated sequences is cleaved by the nutrient-responsive glycosyltransferase, O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT). We report that the tetratricopeptide-repeat domain of O-GlcNAc transferase binds the carboxyl-terminal portion of an HCF-1 proteolytic repeat such that the cleavage region lies in the glycosyltransferase active site above uridine diphosphate-GlcNAc. The conformation is similar to that of a glycosylation-competent peptide substrate. Cleavage occurs between cysteine and glutamate residues and results in a pyroglutamate product. Conversion of the cleavage site glutamate into serine converts an HCF-1 proteolytic repeat into a glycosylation substrate. Thus, protein glycosylation and HCF-1 cleavage occur in the same active site.
Asunto(s)
Factor C1 de la Célula Huésped/química , Factor C1 de la Célula Huésped/metabolismo , N-Acetilglucosaminiltransferasas/química , N-Acetilglucosaminiltransferasas/metabolismo , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Glicosilación , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Conformación Proteica , Estructura Terciaria de Proteína , Proteolisis , Ácido Pirrolidona Carboxílico/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Uridina Difosfato N-Acetilglucosamina/química , Uridina Difosfato N-Acetilglucosamina/metabolismoRESUMEN
Visualization of the reaction coordinate undertaken by glycosyltransferases has remained elusive but is critical for understanding this important class of enzyme. Using substrates and substrate mimics, we describe structural snapshots of all species along the kinetic pathway for human O-linked ß-N-acetylglucosamine transferase (O-GlcNAc transferase), an intracellular enzyme that catalyzes installation of a dynamic post-translational modification. The structures reveal key features of the mechanism and show that substrate participation is important during catalysis.