RESUMO
Tyrosine phosphorylation is a common protein post-translational modification that plays a critical role in signal transduction and the regulation of many cellular processes. Using a propeptide strategy to increase cellular uptake of O-phosphotyrosine (pTyr) and its nonhydrolyzable analog 4-phosphomethyl-L-phenylalanine (Pmp), we identified an orthogonal aminoacyl-tRNA synthetase-tRNA pair that allows site-specific incorporation of both pTyr and Pmp into recombinant proteins in response to the amber stop codon in Escherichia coli in good yields. The X-ray structure of the synthetase reveals a reconfigured substrate-binding site, formed by nonconservative mutations and substantial local structural perturbations. We demonstrate the utility of this method by introducing Pmp into a putative phosphorylation site and determining the affinities of the individual variants for the substrate 3BP2. In summary, this work provides a useful recombinant tool to dissect the biological functions of tyrosine phosphorylation at specific sites in the proteome.
Assuntos
Códon sem Sentido/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Fosfotirosina/análogos & derivados , Fosfotirosina/genética , Cristalografia por Raios X , Ligases/química , Ligases/metabolismo , Modelos Moleculares , Estrutura Molecular , Fosforilação , Fosfotirosina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Disulfide bonds play an important role in protein folding and stability. However, the cross-linking of sites within proteins by cysteine disulfides has significant distance and dihedral angle constraints. Here we report the genetic encoding of noncanonical amino acids containing long side-chain thiols that are readily incorporated into both bacterial and mammalian proteins in good yields and with excellent fidelity. These amino acids can pair with cysteines to afford extended disulfide bonds and allow cross-linking of more distant sites and distinct domains of proteins. To demonstrate this notion, we preformed growth-based selection experiments at nonpermissive temperatures using a library of random ß-lactamase mutants containing these noncanonical amino acids. A mutant enzyme that is cross-linked by one such extended disulfide bond and is stabilized by â¼9 °C was identified. This result indicates that an expanded set of building blocks beyond the canonical 20 amino acids can lead to proteins with improved properties by unique mechanisms, distinct from those possible through conventional mutagenesis schemes.
Assuntos
Dissulfetos , Proteínas de Escherichia coli , Escherichia coli , Dobramento de Proteína , beta-Lactamases , Dissulfetos/química , Dissulfetos/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Estabilidade Proteica , beta-Lactamases/química , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
Thiopeptides are a subclass of ribosomally synthesized and posttranslationally modified peptides (RiPPs) with complex molecular architectures and an array of biological activities, including potent antimicrobial activity. Here we report the generation of thiopeptides containing noncanonical amino acids (ncAAs) by introducing orthogonal amber suppressor aminoacyl-tRNA synthetase/tRNA pairs into a thiocillin producer strain of Bacillus cereus .We demonstrate that thiopeptide variants containing ncAAs with bioorthogonal chemical reactivity can be further postbiosynthetically modified with biophysical probes, including fluorophores and photo-cross-linkers. This work allows the site-specific incorporation of ncAAs into thiopeptides to increase their structural diversity and probe their biological activity; similar approaches can likely be applied to other classes of RiPPs.
Assuntos
Aminoácidos/química , Peptídeos/química , Substituição de Aminoácidos , Aminoácidos/genética , Aminoácidos/metabolismo , Bacillus cereus/genética , Bacillus cereus/metabolismo , Estrutura Molecular , Mutagênese Sítio-Dirigida , Peptídeos/genética , Peptídeos/metabolismo , Engenharia de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Massas em TandemRESUMO
Almost five decades ago Crick, Orgel, and others proposed the RNA world hypothesis. Subsequent studies have raised the possibility that RNA might be able to support both genotype and phenotype, and the function of RNA templates has been studied in terms of evolution, replication, and catalysis. Recently, we engineered strains of E. coli in which a large fraction of 2'-deoxycytidine in the genome is substituted with the modified base 5-hydroxymethyl-2'-deoxycytidine. We now report the generation of mutant strains derived from these engineered bacteria that show significant (â¼40-50%) ribonucleotide content in their genome. We have begun to characterize the properties of these chimeric genomes and the corresponding strains to determine the circumstances under which E. coli can incorporate ribonucleotides into its genome and herein report our initial observations.
Assuntos
DNA Bacteriano/genética , Escherichia coli/genética , Genoma Bacteriano/genética , RNA Bacteriano/genética , Sequência de Bases , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Desoxicitidina/genética , Estrutura MolecularRESUMO
All known living organisms use at least 20 amino acids as the basic building blocks of life. Efforts to reduce the number of building blocks in a replicating system to below the 20 canonical amino acids have not been successful to date. In this work, we use filamentous phage as a model system to investigate the feasibility of removing methionine (Met) from the proteome. We show that all 24 elongation Met sites in the M13 phage genome can be replaced by other canonical amino acids. Most of these changes involve substitution of methionine by leucine (Leu), but in some cases additional compensatory mutations are required. Combining Met substituted sites in the proteome generally led to lower viability/infectivity of the mutant phages, which remains the major challenge in eliminating all methionines from the phage proteome. To date a total of 15 (out of all 24) elongation Mets have been simultaneously deleted from the M13 proteome, providing a useful foundation for future efforts to minimize the genetic code.
Assuntos
Bacteriófago M13/genética , Código Genético/genética , Substituição de Aminoácidos , Bacteriófago M13/metabolismo , Códon , Genoma Viral , Leucina/metabolismo , Metionina/metabolismo , Proteoma/metabolismoRESUMO
With few exceptions, all living organisms encode the same 20 canonical amino acids; however, it remains an open question whether organisms with additional amino acids beyond the common 20 might have an evolutionary advantage. Here, we begin to test that notion by making a large library of mutant enzymes in which 10 structurally distinct noncanonical amino acids were substituted at single sites randomly throughout TEM-1 ß-lactamase. A screen for growth on the ß-lactam antibiotic cephalexin afforded a unique p-acrylamido-phenylalanine (AcrF) mutation at Val-216 that leads to an increase in catalytic efficiency by increasing kcat, but not significantly affecting KM. To understand the structural basis for this enhanced activity, we solved the X-ray crystal structures of the ligand-free mutant enzyme and of the deacylation-defective wild-type and mutant cephalexin acyl-enzyme intermediates. These structures show that the Val-216-AcrF mutation leads to conformational changes in key active site residues-both in the free enzyme and upon formation of the acyl-enzyme intermediate-that lower the free energy of activation of the substrate transacylation reaction. The functional changes induced by this mutation could not be reproduced by substitution of any of the 20 canonical amino acids for Val-216, indicating that an expanded genetic code may offer novel solutions to proteins as they evolve new activities.
Assuntos
Aminoácidos/genética , Evolução Molecular , Código Genético/genética , Modelos Moleculares , beta-Lactamases/química , beta-Lactamases/metabolismo , Catálise , Cefalexina/metabolismo , Clonagem Molecular , Cristalização , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Vetores Genéticos/genética , Cinética , Estrutura Molecular , Conformação Proteica , Difração de Raios X , beta-Lactamases/genéticaRESUMO
Several modified bases have been observed in the genomic DNA of bacteriophages, prokaryotes, and eukaryotes that play a role in restriction systems and/or epigenetic regulation. In our efforts to understand the consequences of replacing a large fraction of a canonical nucleoside with a modified nucleoside, we previously replaced around 75% of thymidine (T) with 5'-hydroxymethyl-2'-deoxyuridine (5hmU) in the Escherichia coli genome. In this study, we engineered the pyrimidine nucleotide biosynthetic pathway using T4 bacteriophage genes to achieve approximately 63% replacement of 2'-deoxycytidine (dC) with 5-hydroxymethyl-2'-deoxycytidine (5hmC) in the E. coli genome and approximately 71% replacement in plasmids. We further engineered the glucose metabolic pathway to transform the 5hmC into glucosyl-5-hydroxymethyl-2'-deoxycytidine (5-gmC) and achieved 20% 5-gmC in the genome and 45% 5-gmC in plasmid DNA.
Assuntos
Desoxicitidina/metabolismo , Escherichia coli/genética , Engenharia Genética , Genoma Bacteriano/genética , Bacteriófago T4/genética , Genes Virais/genéticaRESUMO
Prokaryotic and eukaryotic genomic DNA is comprised of the four building blocks A, G, C, and T. We have begun to explore the consequences of replacing a large fraction or all of a nucleoside in genomic DNA with a modified nucleoside. As a first step we have investigated the possibility of replacement of T by 2'-deoxy-5-(hydroxymethyl)uridine (5hmU) in the genomic DNA of Escherichia coli. Metabolic engineering with phage genes followed by random mutagenesis enabled us to achieve approximately 75% replacement of T by 5hmU in the E. coli genome and in plasmids.
Assuntos
Escherichia coli/genética , Engenharia Genética/métodos , Genoma Bacteriano , Timidina/análogos & derivados , Timidina/genética , Sequência de Bases , DNA Bacteriano/genética , Plasmídeos/genéticaRESUMO
The first general strategy for a directing effect on metal (oxo)-promoted C-H hydroxylations is described. Carboxylic acid moieties on the substrate overcome unfavorable electronic, steric, and stereoelectronic biases in C-H hydroxylations catalyzed by the non-heme iron complex Fe(PDP). In a demonstration of the power of this directing effect, C-H oxidation is diverted away from an electronically favored C-1 H abstraction/rearrangement pathway in the paclitaxel framework to enable installation of C-2 oxidation in the naturally occurring oxidation state and stereoconfiguration.
Assuntos
Ácidos Carboxílicos/química , Compostos Ferrosos/química , Hidrocarbonetos Aromáticos com Pontes/química , Catálise , Elétrons , Hidroxilação , Oxirredução , Estereoisomerismo , Taxoides/químicaRESUMO
A Brønsted base activation mode for oxidative, Pd(II)/sulfoxide-catalyzed, intermolecular C-H allylic amination is reported. N,N-diisopropylethylamine was found to promote amination of unactivated terminal olefins, forming the corresponding linear allylic amine products with high levels of stereo-, regio-, and chemoselectivity. The predictable and high selectivity of this C-H oxidation method enables late-stage incorporation of nitrogen into advanced synthetic intermediates and natural products.
Assuntos
Carbono/química , Hidrogênio/química , Aminação , Produtos Biológicos/química , Carbamatos/química , Catálise , Concentração de Íons de Hidrogênio , Oxirredução , Paládio/químicaRESUMO
Over 2,000 human proteins are predicted to be secreted, but the biological function of the many of these proteins is still unknown. Moreover, a number of these proteins may act as new therapeutic agents or be targets for the development of therapeutic antibodies. To further explore the extracellular proteome, we have developed a secretome-enriched open reading frame (ORF) library that can be readily screened for autocrine activity in cell-based phenotypic or reporter assays. Next-generation sequencing (NGS) and database analysis predict that the library contains approximately 900 ORFs encoding known secreted proteins (accounting for 77.8% of the library), as well as genes encoding potentially unknown secreted proteins. In a proof-of-principle study, human TF-1 cells were screened for proliferative factors, and the known cytokine GMCSF was identified as a dominant hit. This library offers a relatively low-cost and straightforward approach for functional autocrine screens of secreted proteins.
Assuntos
Biblioteca Gênica , Lentivirus/genética , Proteoma/genética , Proteoma/metabolismo , Sequência de Aminoácidos , Fases de Leitura Aberta/genética , Fenótipo , Proteoma/químicaRESUMO
Carboxylate-ligated, non-haem iron enzymes demonstrate the capacity for catalysing such remarkable processes as hydroxylations, chlorinations and desaturations of inert, aliphatic C-H bonds. A key to functional diversity is the enzymes' ability to divert fleeting radicals towards different types of functionalization using active site and/or substrate modifications. We report that a non-haem iron hydroxylase catalyst [Fe(PDP)] can also be diverted to catalytic, mixed hydroxylase/desaturase activity with aliphatic C-H bonds. Using a taxane-based radical trap that rearranges under Fe(PDP) oxidation to furnish a nortaxane skeleton, we provide the first direct evidence for a substrate radical using this class of stereoretentive hydroxylation catalysts. Hydroxylation and desaturation proceed by means of a short-lived radical that diverges in a substrate-dependent manner in the presence of carboxylic acids. The novel biomimetic reactivity displayed by this small molecule catalyst is harnessed to diversify natural product derivatives as well as interrogate their biosynthetic pathways.