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1.
Sci Rep ; 12(1): 16991, 2022 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-36216916

RESUMO

N-acetylglucosamine (GlcNAc) is a key component of glycans such as glycoprotein and the cell wall. GlcNAc kinase is an enzyme that transfers a phosphate onto GlcNAc to generate GlcNAc-6-phosphate, which can be a precursor for glycan synthesis. GlcNAc kinases have been found in a broad range of organisms, including pathogenic yeast, human and bacteria. However, this enzyme has never been discovered in Saccharomyces cerevisiae, a eukaryotic model. In this study, the first GlcNAc kinase from S. cerevisiae was identified and named Ngk1. The Km values of Ngk1 for GlcNAc and glucose were 0.11 mM and 71 mM, respectively, suggesting that Ngk1 possesses a high affinity for GlcNAc, unlike hexokinases. Ngk1 showed the GlcNAc phosphorylation activity with various nucleoside triphosphates, namely ATP, CTP, GTP, ITP, and UTP, as phosphoryl donors. Ngk1 is phylogenetically distant from known enzymes, as the amino acid sequence identity with others is only about 20% or less. The physiological role of Ngk1 in S. cerevisiae is also discussed.


Assuntos
Acetilglucosamina , Fosfotransferases (Aceptor do Grupo Álcool) , Saccharomyces cerevisiae , Acetilglucosamina/metabolismo , Trifosfato de Adenosina/metabolismo , Citidina Trifosfato/metabolismo , Glucose/metabolismo , Glicoproteínas/metabolismo , Guanosina Trifosfato/metabolismo , Nucleosídeos/metabolismo , Fosfatos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Polissacarídeos/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Uridina Trifosfato/metabolismo
2.
Sci Rep ; 12(1): 18084, 2022 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-36302925

RESUMO

Hexokinase 2 (Hxk2) of Saccharomyces cerevisiae is a dual function hexokinase, acting as a glycolytic enzyme and being involved in the transcriptional regulation of glucose-repressible genes. Relief from glucose repression is accompanied by phosphorylation of Hxk2 at serine 15, which has been attributed to the protein kinase Tda1. To explore the role of Tda1 beyond Hxk2 phosphorylation, the proteomic consequences of TDA1 deficiency were investigated by difference gel electrophoresis (2D-DIGE) comparing a wild type and a Δtda1 deletion mutant. To additionally address possible consequences of glucose repression/derepression, both were grown at 2% and 0.1% (w/v) glucose. A total of eight protein spots exhibiting a minimum twofold enhanced or reduced fluorescence upon TDA1 deficiency was detected and identified by mass spectrometry. Among the spot identities are-besides the expected Hxk2-two proteoforms of hexokinase 1 (Hxk1). Targeted proteomics analyses in conjunction with 2D-DIGE demonstrated that TDA1 is indispensable for Hxk2 and Hxk1 phosphorylation at serine 15. Thirty-six glucose-concentration-dependent protein spots were identified. A simple method to improve spot quantification, approximating spots as rotationally symmetric solids, is presented along with new data on the quantities of Hxk1 and Hxk2 and their serine 15 phosphorylated forms at high and low glucose growth conditions. The Δtda1 deletion mutant exhibited no altered growth under high or low glucose conditions or on alternative carbon sources. Also, invertase activity, serving as a reporter for glucose derepression, was not significantly altered. Instead, an involvement of Tda1 in oxidative stress response is suggested.


Assuntos
Hexoquinase , Proteínas Quinases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Hexoquinase/genética , Hexoquinase/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Proteômica , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/metabolismo
3.
Proc Natl Acad Sci U S A ; 119(33): e2205848119, 2022 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-35939674

RESUMO

Tetrahydropapaverine (THP) and papaverine are plant natural products with clinically significant roles. THP is a precursor in the production of the drugs atracurium and cisatracurium, and papaverine is used as an antispasmodic during vascular surgery. In recent years, metabolic engineering advances have enabled the production of natural products through heterologous expression of pathway enzymes in yeast. Heterologous biosynthesis of THP and papaverine could play a role in ensuring a stable supply of these clinically significant products. Biosynthesis of THP and papaverine has not been achieved to date, in part because multiple pathway enzymes have not been elucidated. Here, we describe the development of an engineered yeast strain for de novo biosynthesis of THP. The production of THP is achieved through heterologous expression of two enzyme variants with activity on nonnative substrates. Through protein engineering, we developed a variant of N-methylcoclaurine hydroxylase with activity on coclaurine, enabling de novo norreticuline biosynthesis. Similarly, we developed a variant of scoulerine 9-O-methyltransferase capable of O-methylating 1-benzylisoquinoline alkaloids at the 3' position, enabling de novo THP biosynthesis. Flux through the heterologous pathway was improved by knocking out yeast multidrug resistance transporters and optimization of media conditions. Overall, strain engineering increased the concentration of biosynthesized THP 600-fold to 121 µg/L. Finally, we demonstrate a strategy for papaverine semisynthesis using hydrogen peroxide as an oxidizing agent. Through optimizing pH, temperature, reaction time, and oxidizing agent concentration, we demonstrated the ability to produce semisynthesized papaverine through oxidation of biosynthesized THP.


Assuntos
Produtos Biológicos , Papaverina , Engenharia de Proteínas , Saccharomyces cerevisiae , Produtos Biológicos/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Peróxido de Hidrogênio/química , Oxidantes/química , Papaverina/biossíntese , Proteínas de Plantas/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética
4.
Nucleic Acids Res ; 50(14): 8023-8040, 2022 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-35822874

RESUMO

Amino acid substitutions in the exonuclease domain of DNA polymerase ϵ (Polϵ) cause ultramutated tumors. Studies in model organisms suggested pathogenic mechanisms distinct from a simple loss of exonuclease. These mechanisms remain unclear for most recurrent Polϵ mutations. Particularly, the highly prevalent V411L variant remained a long-standing puzzle with no detectable mutator effect in yeast despite the unequivocal association with ultramutation in cancers. Using purified four-subunit yeast Polϵ, we assessed the consequences of substitutions mimicking human V411L, S459F, F367S, L424V and D275V. While the effects on exonuclease activity vary widely, all common cancer-associated variants have increased DNA polymerase activity. Notably, the analog of Polϵ-V411L is among the strongest polymerases, and structural analysis suggests defective polymerase-to-exonuclease site switching. We further show that the V411L analog produces a robust mutator phenotype in strains that lack mismatch repair, indicating a high rate of replication errors. Lastly, unlike wild-type and exonuclease-dead Polϵ, hyperactive variants efficiently synthesize DNA at low dNTP concentrations. We propose that this characteristic could promote cancer cell survival and preferential participation of mutator polymerases in replication during metabolic stress. Our results support the notion that polymerase fitness, rather than low fidelity alone, is an important determinant of variant pathogenicity.


Assuntos
DNA Polimerase II , Neoplasias , Nucleotídeos , Proteínas de Saccharomyces cerevisiae , DNA Polimerase II/metabolismo , Replicação do DNA/genética , Exonucleases/genética , Humanos , Mutação , Neoplasias/enzimologia , Neoplasias/genética , Nucleotídeos/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
Biochemistry ; 61(13): 1273-1285, 2022 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-35730892

RESUMO

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a eukaryotic, post-translational modification catalyzed by GPI transamidase (GPI-T). The Saccharomyces cerevisiae GPI-T is composed of five membrane-bound subunits: Gpi8, Gaa1, Gpi16, Gpi17, and Gab1. GPI-T has been recalcitrant to in vitro structure and function studies because of its complexity and membrane-solubility. Furthermore, a reliable, quantitative, in vitro assay for this important post-translational modification has remained elusive despite its discovery more than three decades ago.Three recent reports describe the structure of GPI-T from S. cerevisiae and humans, shedding critical light on this important enzyme and offering insight into the functions of its different subunits. Here, we present the purification and characterization of a truncated soluble GPI-T heterotrimer complex (Gpi823-306, Gaa150-343, and Gpi1620-551) without transmembrane domains. Using this simplified heterotrimer, we report the first quantitative method to measure GPI-T activity in vitro and demonstrate that this soluble, minimalistic GPI-T retains transamidase activity. These results contribute to our understanding of how this enzyme is organized and functions, and provide a method to screen potential GPI-T inhibitors.


Assuntos
Aciltransferases , Proteínas de Saccharomyces cerevisiae , Aciltransferases/química , Aciltransferases/metabolismo , Glicosilfosfatidilinositóis , Humanos , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo
6.
J Cell Biol ; 221(6)2022 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-35587358

RESUMO

The guided entry of tail-anchored proteins (GET) pathway targets C-terminally anchored transmembrane proteins and protects cells from lipotoxicity. Here, we reveal perturbed ergosterol production in ∆get3 cells and demonstrate the sensitivity of GET pathway mutants to the sterol synthesis inhibiting drug terbinafine. Our data uncover a key enzyme of sterol synthesis, the hairpin membrane protein squalene monooxygenase (Erg1), as a non-canonical GET pathway client, thus rationalizing the lipotoxicity phenotypes of GET pathway mutants. Get3 recognizes the hairpin targeting element of Erg1 via its classical client-binding pocket. Intriguingly, we find that the GET pathway is especially important for the acute upregulation of Erg1 induced by low sterol conditions. We further identify several other proteins anchored to the endoplasmic reticulum (ER) membrane exclusively via a hairpin as putative clients of the GET pathway. Our findings emphasize the necessity of dedicated targeting pathways for high-efficiency targeting of particular clients during dynamic cellular adaptation and highlight hairpin proteins as a potential novel class of GET clients.


Assuntos
Adenosina Trifosfatases , Fatores de Troca do Nucleotídeo Guanina , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Esqualeno Mono-Oxigenase , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Retículo Endoplasmático/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Esqualeno Mono-Oxigenase/genética , Esqualeno Mono-Oxigenase/metabolismo , Esteróis/metabolismo
7.
Genetics ; 221(4)2022 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-35608294

RESUMO

Acetyl-CoA Carboxylase 1 catalyzes the conversion of acetyl-CoA to malonyl-CoA, the committed step of de novo fatty acid synthesis. As a master regulator of lipid synthesis, acetyl-CoA carboxylase 1 has been proposed to be a therapeutic target for numerous metabolic diseases. We have shown that acetyl-CoA carboxylase 1 activity is reduced in the absence of the lysine acetyltransferase NuA4 in Saccharomyces cerevisiae. This change in acetyl-CoA carboxylase 1 activity is correlated with a change in localization. In wild-type cells, acetyl-CoA carboxylase 1 is localized throughout the cytoplasm in small punctate and rod-like structures. However, in NuA4 mutants, acetyl-CoA carboxylase 1 localization becomes diffuse. To uncover mechanisms regulating acetyl-CoA carboxylase 1 localization, we performed a microscopy screen to identify other deletion mutants that impact acetyl-CoA carboxylase 1 localization and then measured acetyl-CoA carboxylase 1 activity in these mutants through chemical genetics and biochemical assays. Three phenotypes were identified. Mutants with hyper-active acetyl-CoA carboxylase 1 form 1 or 2 rod-like structures centrally within the cytoplasm, mutants with mid-low acetyl-CoA carboxylase 1 activity displayed diffuse acetyl-CoA carboxylase 1, while the mutants with the lowest acetyl-CoA carboxylase 1 activity (hypomorphs) formed thick rod-like acetyl-CoA carboxylase 1 structures at the periphery of the cell. All the acetyl-CoA carboxylase 1 hypomorphic mutants were implicated in sphingolipid metabolism or very long-chain fatty acid elongation and in common, their deletion causes an accumulation of palmitoyl-CoA. Through exogenous lipid treatments, enzyme inhibitors, and genetics, we determined that increasing palmitoyl-CoA levels inhibits acetyl-CoA carboxylase 1 activity and remodels acetyl-CoA carboxylase 1 localization. Together this study suggests yeast cells have developed a dynamic feed-back mechanism in which downstream products of acetyl-CoA carboxylase 1 can fine-tune the rate of fatty acid synthesis.


Assuntos
Acetil-CoA Carboxilase , Histona Acetiltransferases , Lisina Acetiltransferases , Proteínas de Saccharomyces cerevisiae , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Coenzima A/metabolismo , Ácidos Graxos/metabolismo , Genômica , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Lipídeos , Lisina Acetiltransferases/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Esfingolipídeos/metabolismo
8.
Microbiol Spectr ; 10(3): e0082222, 2022 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-35543513

RESUMO

In the yeast Saccharomyces cerevisiae, N-acetyl glutamate kinase (NAGK), which catalyzes the phosphorylation of N-acetyl glutamate to form N-acetyl glutamyl-5-phosphate, is one of the rate-limiting enzymes in the ornithine and arginine biosynthetic pathways. NAGK activity is strictly regulated via feedback inhibition by the end product, arginine. We previously reported that the Thr340Ile variant of NAGK was insensitive to arginine feedback inhibition and that the interaction between Lys336 and Thr340 in NAGK may be important for arginine recognition. In the present study, we demonstrated that amino acid changes of Thr340 to Ala, Leu, Arg, Glu, Ile, and Asn removed arginine feedback inhibition, although the Thr340Ser variant was subject to the feedback inhibition. Therefore, these results indicate that the arginine-binding cavity formed via the interaction between the carbonyl group in the main chain of Lys336 and the hydroxyl group in the side chain of the residue at position 340 is critical for arginine recognition of NAGK. In addition, we newly identified two mutations in the ARG5,6 gene encoding the Cys119Tyr or Val267Ala variant of NAGK of sake yeast mutants with intracellular ornithine accumulation. Although it is unlikely that Cys119 and Val267 are directly involved in arginine recognition, we found here that two variants of NAGK were insensitive to arginine feedback inhibition and contributed to high-level production of ornithine. Structural analysis of NAGK suggests that these two amino acid substitutions influence the sensitivity to Arg feedback inhibition through alterations in local conformation around each residue. IMPORTANCE Ornithine has a number of physiological benefits in humans. Thus, an Orn-rich alcoholic beverage is expected to relieve feelings of fatigue after drinking. In the yeast Saccharomyces cerevisiae, N-acetyl glutamate kinase (NAGK) encoded by the ARG5,6 gene catalyzes the second step in ornithine and arginine biosynthesis, and its activity is subjected to feedback inhibition by arginine. Here, we revealed a role of key residues in the formation of the arginine-binding cavity which is critical for arginine recognition of NAGK. In addition, we analyzed novel arginine feedback inhibition-insensitive variants of NAGK in sake yeast mutants with ornithine overproduction and proposed that the amino acid substitutions in the NAGK variants destabilize the arginine-binding cavity, leading to the lower sensitivity to arginine feedback inhibition of NAGK activity. These findings provide new insight into the allosteric regulation of NAGK activity and will help to construct superior industrial yeast strains for high-level production of ornithine.


Assuntos
Ornitina , Fosfotransferases (Aceptor do Grupo Carboxila) , Saccharomyces cerevisiae , Bebidas Alcoólicas , Arginina/química , Retroalimentação , Ornitina/biossíntese , Fosfotransferases (Aceptor do Grupo Carboxila)/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética
9.
Methods ; 204: 234-240, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35483549

RESUMO

DNA helicases function in many types of nucleic acid transactions, and as such, they are vital for genome integrity. Although they are often considered individually, work from many groups demonstrates that these enzymes often genetically and biochemically interact in vivo. Here, we highlight methods to interrogate such interactions among the PIF1 (Pif1 and Rrm3) and RecQ (Hrq1 and Sgs1) family helicases in Saccharomyces cerevisiae. The interactions among these enzymes were investigated in vivo using deletion and inactivation alleles with a gross-chromosomal rearrangement (GCR) assay. Further, wild-type and inactive recombinant proteins were used to determine the effects of the helicases on telomerase activity in vitro. We found that synergistic increases in GCR rates often occur in double vs. single mutants, suggesting that the helicases function in distinct genome integrity pathways. Further, the recombinant helicases can function together in vitro to modulate telomerase activity. Overall, the data suggest that the interactions among the members of these DNA helicase families are multipartite and argue for a comprehensive systems biology approach to fully elucidate the physiological interplay between these enzymes.


Assuntos
DNA Helicases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Telomerase , DNA Helicases/genética , DNA Helicases/metabolismo , RecQ Helicases/genética , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Telomerase/metabolismo
10.
J Mol Biol ; 434(17): 167531, 2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-35259366

RESUMO

Proteins fluctuate between different conformations in solution, and these conformational fluctuations can be important for protein function and allosteric regulation. The chorismate mutase from Saccharomyces cerevisiae (ScCM), a key enzyme in the biosynthesis of aromatic amino acids, is allosterically activated and inhibited by tryptophan and tyrosine, respectively. It was initially proposed that in the absence of effector, ScCM fluctuates between activated R and inhibited T conformations according to the Monod-Wyman-Changeux (MWC) model, although a more complex regulation pattern was later suggested by mutagenesis and kinetic data. Here we used NMR relaxation dispersion experiments to understand the conformational fluctuations on the microsecond-to-millisecond timescale that occur in ScCM. In the absence of allosteric effectors, ScCM did not exclusively exchange between T and R conformations, suggesting that the two-state MWC model is insufficient to explain conformational dynamics. Addition of tyrosine led to the quenching of much of the motion on this timescale, while new motions were identified in the presence of tryptophan. These new motions are consistent with conformational fluctuations into an alternative conformation that may be important for enzyme activity.


Assuntos
Corismato Mutase , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Regulação Alostérica , Corismato Mutase/química , Espectroscopia de Ressonância Magnética , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Triptofano/metabolismo , Tirosina/metabolismo
11.
Mol Cell ; 82(11): 2006-2020.e8, 2022 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-35353987

RESUMO

CK1s are acidophilic serine/threonine kinases with multiple critical cellular functions; their misregulation contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Here, we describe an evolutionarily conserved mechanism of CK1 activity: autophosphorylation of a threonine (T220 in human CK1δ) located at the N terminus of helix αG, proximal to the substrate binding cleft. Crystal structures and molecular dynamics simulations uncovered inherent plasticity in αG that increased upon T220 autophosphorylation. The phosphorylation-induced structural changes significantly altered the conformation of the substrate binding cleft, affecting substrate specificity. In T220 phosphorylated yeast and human CK1s, activity toward many substrates was decreased, but we also identified a high-affinity substrate that was phosphorylated more rapidly, and quantitative phosphoproteomics revealed that disrupting T220 autophosphorylation rewired CK1 signaling in Schizosaccharomyces pombe. T220 is present exclusively in the CK1 family, thus its autophosphorylation may have evolved as a unique regulatory mechanism for this important family.


Assuntos
Proteínas Serina-Treonina Quinases , Caseína Quinase Idelta , Humanos , Fosforilação , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Transdução de Sinais , Especificidade por Substrato , Treonina
12.
J Enzyme Inhib Med Chem ; 37(1): 554-562, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35152818

RESUMO

Aiming at finding natural sources of antidiabetics agents, 15 extracts from Brazilian medicinal plants of the Atlantic Forest and Amazon region were tested against α-glucosidase enzyme. Plants were selected based on the taxonomic relationships with genera including several species with antidiabetic activity. In this screening, the extracts obtained from the flowers of Hyptis monticola and the leaves of Lantana trifolia and Lippia origanoides resulted endowed with promising anti-α-glucosidase activity. The extracts from H. monticola and from L. origanoides collected in two different areas, were characterised by ultra-high performance liquid chromatography coupled to mass spectrometry. Bioassay-guided fractionation led to the identification of several enzyme inhibiting compounds, among them the mechanism of action of naringenin and pinocembrin was investigated. The two L. origanoides extracts showed differences in bioactivity and in the phytochemical profiles. The fractionation of the extract from H. monticola led to a partial loss of the inhibitory effect.


Assuntos
Inibidores de Glicosídeo Hidrolases/farmacologia , Hyptis/química , Lantana/química , Extratos Vegetais/farmacologia , Plantas Medicinais/química , alfa-Glucosidases/metabolismo , Brasil , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Flores/química , Inibidores de Glicosídeo Hidrolases/química , Inibidores de Glicosídeo Hidrolases/isolamento & purificação , Espectrometria de Massas , Estrutura Molecular , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Folhas de Planta/química , Saccharomyces cerevisiae/enzimologia , Relação Estrutura-Atividade
13.
J Mol Biol ; 434(7): 167478, 2022 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-35123996

RESUMO

Despite decades of research and the availability of the full genomic sequence of the baker's yeast Saccharomyces cerevisiae, still a large fraction of its genome is not functionally annotated. This hinders our ability to fully understand cellular activity and suggests that many additional processes await discovery. The recent years have shown an explosion of high-quality genomic and structural data from multiple organisms, ranging from bacteria to mammals. New computational methods now allow us to integrate these data and extract meaningful insights into the functional identity of uncharacterized proteins in yeast. Here, we created a database of sensitive sequence similarity predictions for all yeast proteins. We use this information to identify candidate enzymes for known biochemical reactions whose enzymes are unidentified, and show how this provides a powerful basis for experimental validation. Using one pathway as a test case we pair a new function for the previously uncharacterized enzyme Yhr202w, as an extra-cellular AMP hydrolase in the NAD degradation pathway. Yhr202w, which we now term Smn1 for Scavenger MonoNucleotidase 1, is a highly conserved protein that is similar to the human protein E5NT/CD73, which is associated with multiple cancers. Hence, our new methodology provides a paradigm, that can be adopted to other organisms, for uncovering new enzymatic functions of uncharacterized proteins.


Assuntos
Monofosfato de Adenosina , Nucleotidases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Monofosfato de Adenosina/química , Humanos , Nucleotidases/química , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Análise de Sequência de Proteína/métodos
14.
Mol Cell ; 82(3): 660-676.e9, 2022 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-35051353

RESUMO

Previous structural studies of the initiation-elongation transition of RNA polymerase II (pol II) transcription have relied on the use of synthetic oligonucleotides, often artificially discontinuous to capture pol II in the initiating state. Here, we report multiple structures of initiation complexes converted de novo from a 33-subunit yeast pre-initiation complex (PIC) through catalytic activities and subsequently stalled at different template positions. We determine that PICs in the initially transcribing complex (ITC) can synthesize a transcript of ∼26 nucleotides before transitioning to an elongation complex (EC) as determined by the loss of general transcription factors (GTFs). Unexpectedly, transition to an EC was greatly accelerated when an ITC encountered a downstream EC stalled at promoter proximal regions and resulted in a collided head-to-end dimeric EC complex. Our structural analysis reveals a dynamic state of TFIIH, the largest of GTFs, in PIC/ITC with distinct functional consequences at multiple steps on the pathway to elongation.


Assuntos
RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Iniciação da Transcrição Genética , Microscopia Crioeletrônica , Regulação Fúngica da Expressão Gênica , Modelos Moleculares , Regiões Promotoras Genéticas , Conformação Proteica , RNA Polimerase II/genética , RNA Polimerase II/ultraestrutura , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Relação Estrutura-Atividade , Fatores de Tempo , Elongação da Transcrição Genética , Fatores de Transcrição TFII/genética , Fatores de Transcrição TFII/metabolismo
15.
Nat Commun ; 13(1): 95, 2022 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-35013177

RESUMO

Non-heme iron and α-ketoglutarate-dependent (Fe/αKG) oxygenases catalyze various oxidative biotransformations. Due to their catalytic flexibility and high efficiency, Fe/αKG oxygenases have attracted keen attention for their application as biocatalysts. Here, we report the biochemical and structural characterizations of the unusually promiscuous and catalytically versatile Fe/αKG oxygenase SptF, involved in the biosynthesis of fungal meroterpenoid emervaridones. The in vitro analysis revealed that SptF catalyzes several continuous oxidation reactions, including hydroxylation, desaturation, epoxidation, and skeletal rearrangement. SptF exhibits extremely broad substrate specificity toward various meroterpenoids, and efficiently produced unique cyclopropane-ring-fused 5/3/5/5/6/6 and 5/3/6/6/6 scaffolds from terretonins. Moreover, SptF also hydroxylates steroids, including androsterone, testosterone, and progesterone, with different regiospecificities. Crystallographic and structure-based mutagenesis studies of SptF revealed the molecular basis of the enzyme reactions, and suggested that the malleability of the loop region contributes to the remarkable substrate promiscuity. SptF exhibits great potential as a promising biocatalyst for oxidation reactions.


Assuntos
Proteínas Fúngicas/química , Ferro/química , Ácidos Cetoglutáricos/química , Oxirredutases N-Desmetilantes/química , Terpenos/química , Androsterona/química , Androsterona/metabolismo , Sítios de Ligação , Biocatálise , Cátions Bivalentes , Cristalografia por Raios X , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Humanos , Hidroxilação , Ferro/metabolismo , Ácidos Cetoglutáricos/metabolismo , Cinética , Modelos Moleculares , Mutação , Oxirredução , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Progesterona/química , Progesterona/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Especificidade por Substrato , Terpenos/classificação , Terpenos/metabolismo , Testosterona/química , Testosterona/metabolismo
16.
Chem Res Toxicol ; 35(2): 326-336, 2022 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-35084835

RESUMO

Protein disulfide isomerases (PDIs) function in forming the correct disulfide bonds in client proteins, thereby aiding the folding of proteins that enter the secretory pathway. Recently, several PDIs have been identified as targets of organic electrophiles, yet the client proteins of specific PDIs remain largely undefined. Here, we report that PDIs expressed in Saccharomyces cerevisiae are targets of divinyl sulfone (DVSF) and other thiol-reactive protein cross-linkers. Using DVSF, we identified the interaction partners that were cross-linked to Pdi1 and Eug1, finding that both proteins form cross-linked complexes with other PDIs, as well as vacuolar hydrolases, proteins involved in cell wall biosynthesis and maintenance, and many ER proteostasis factors involved ER stress signaling and ER-associated protein degradation (ERAD). The latter discovery prompted us to examine the effects of DVSF on ER quality control, where we found that DVSF inhibits the degradation of the ERAD substrate CPY*, in addition to covalently modifying Ire1 and blocking the activation of the unfolded protein response. Our results reveal that DVSF targets many proteins within the ER proteostasis network and suggest that these proteins may be suitable targets for covalent therapeutic development in the future.


Assuntos
Reagentes de Ligações Cruzadas/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Saccharomyces cerevisiae/enzimologia , Compostos de Sulfidrila/metabolismo , Reagentes de Ligações Cruzadas/química , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Estrutura Molecular , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/química , Proteólise/efeitos dos fármacos , Proteostase/efeitos dos fármacos , Compostos de Sulfidrila/química , Sulfonas/farmacologia
17.
J Med Chem ; 65(3): 2471-2496, 2022 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-35077178

RESUMO

Novel analogues of C-2-substituted thienopyrimidine-based bisphosphonates (C2-ThP-BPs) are described that are potent inhibitors of the human geranylgeranyl pyrophosphate synthase (hGGPPS). Members of this class of compounds induce target-selective apoptosis of multiple myeloma (MM) cells and exhibit antimyeloma activity in vivo. A key structural element of these inhibitors is a linker moiety that connects their (((2-phenylthieno[2,3-d]pyrimidin-4-yl)amino)methylene)bisphosphonic acid core to various side chains. The structural diversity of this linker moiety, as well as the side chains attached to it, was investigated and found to significantly impact the toxicity of these compounds in MM cells. The most potent inhibitor identified was evaluated in mouse and rat for liver toxicity and systemic exposure, respectively, providing further optimism for the potential value of such compounds as human therapeutics.


Assuntos
Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Geranil-Geranildifosfato Geranil-Geraniltransferase/antagonistas & inibidores , Mieloma Múltiplo/tratamento farmacológico , Pirimidinas/uso terapêutico , Tiofenos/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/toxicidade , Células da Medula Óssea/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/toxicidade , Feminino , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/metabolismo , Geranil-Geranildifosfato Geranil-Geraniltransferase/metabolismo , Humanos , Fígado/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Estrutura Molecular , Ligação Proteica , Pirimidinas/síntese química , Pirimidinas/metabolismo , Pirimidinas/toxicidade , Ratos , Saccharomyces cerevisiae/enzimologia , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/metabolismo , Tiofenos/toxicidade
18.
Sci Rep ; 12(1): 405, 2022 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-35013392

RESUMO

The carbohydrate-hydrolyzing enzymes play a crucial role in increasing the phenolic content and nutritional properties of polysaccharides substrate, essential for cost-effective industrial applications. Also, improving the feed efficiency of poultry is essential to achieve significant economic benefits. The current study introduced a novel thermostable metagenome-derived xylanase named PersiXyn8 and investigated its synergistic effect with previously reported α-amylase (PersiAmy3) to enhance poultry feed utilization. The potential of the enzyme cocktail in the degradation of poultry feed was analyzed and showed 346.73 mg/g poultry feed reducing sugar after 72 h of hydrolysis. Next, the impact of solid-state fermentation on corn quality was investigated in the presence and absence of enzymes. The phenolic content increased from 36.60 mg/g GAE in control sample to 68.23 mg/g in the presence of enzymes. In addition, the enzyme-treated sample showed the highest reducing power OD 700 of 0.217 and the most potent radical scavenging activity against ABTS (40.36%) and DPPH (45.21%) radicals. Moreover, the protein and ash contents of the fermented corn increased by 4.88% and 6.46%, respectively. These results confirmed the potential of the carbohydrate-hydrolyzing enzymes cocktail as a low-cost treatment for improving the phenolic content, antioxidant activity, and nutritional values of corn for supplementation of corn-based poultry feed.


Assuntos
Ração Animal , Manipulação de Alimentos , Valor Nutritivo , Aves Domésticas , Xilosidases/metabolismo , Zea mays/metabolismo , alfa-Amilases/metabolismo , Animais , Fermentação , Hidrólise , Fenóis/metabolismo , Saccharomyces cerevisiae/enzimologia , Especificidade por Substrato , Açúcares/metabolismo , Xilosidases/genética , Zea mays/microbiologia
19.
J Biol Chem ; 298(2): 101570, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35026224

RESUMO

In mitochondria, cysteine desulfurase (Nfs1) plays a central role in the biosynthesis of iron-sulfur (FeS) clusters, cofactors critical for activity of many cellular proteins. Nfs1 functions both as a sulfur donor for cluster assembly and as a binding platform for other proteins functioning in the process. These include not only the dedicated scaffold protein (Isu1) on which FeS clusters are synthesized but also accessory FeS cluster biogenesis proteins frataxin (Yfh1) and ferredoxin (Yah1). Yfh1 has been shown to activate cysteine desulfurase enzymatic activity, whereas Yah1 supplies electrons for the persulfide reduction. While Yfh1 interaction with Nfs1 is well understood, the Yah1-Nfs1 interaction is not. Here, based on the results of biochemical experiments involving purified WT and variant proteins, we report that in Saccharomyces cerevisiae, Yah1 and Yfh1 share an evolutionary conserved interaction site on Nfs1. Consistent with this notion, Yah1 and Yfh1 can each displace the other from Nfs1 but are inefficient competitors when a variant with an altered interaction site is used. Thus, the binding mode of Yah1 and Yfh1 interacting with Nfs1 in mitochondria of S. cerevisiae resembles the mutually exclusive binding of ferredoxin and frataxin with cysteine desulfurase reported for the bacterial FeS cluster assembly system. Our findings are consistent with the generally accepted scenario that the mitochondrial FeS cluster assembly system was inherited from bacterial ancestors of mitochondria.


Assuntos
Ferredoxinas , Proteínas Ferro-Enxofre , Proteínas Mitocondriais , Proteínas de Saccharomyces cerevisiae , Sulfurtransferases , Sítios de Ligação , Liases de Carbono-Enxofre/genética , Liases de Carbono-Enxofre/metabolismo , Ferredoxinas/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Proteínas Mitocondriais/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sulfurtransferases/metabolismo
20.
Biomolecules ; 12(1)2022 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-35053213

RESUMO

Genome-scale metabolic models (GEMs) have been widely used for the phenotypic prediction of microorganisms. However, the lack of other constraints in the stoichiometric model often leads to a large metabolic solution space being inaccessible. Inspired by previous studies that take an allocation of macromolecule resources into account, we developed a simplified Python-based workflow for constructing enzymatic constrained metabolic network model (ECMpy) and constructed an enzyme-constrained model for Escherichia coli (eciML1515) by directly adding a total enzyme amount constraint in the latest version of GEM for E. coli (iML1515), considering the protein subunit composition in the reaction, and automated calibration of enzyme kinetic parameters. Using eciML1515, we predicted the overflow metabolism of E. coli and revealed that redox balance was the key reason for the difference between E. coli and Saccharomyces cerevisiae in overflow metabolism. The growth rate predictions on 24 single-carbon sources were improved significantly when compared with other enzyme-constrained models of E. coli. Finally, we revealed the tradeoff between enzyme usage efficiency and biomass yield by exploring the metabolic behaviours under different substrate consumption rates. Enzyme-constrained models can improve simulation accuracy and thus can predict cellular phenotypes under various genetic perturbations more precisely, providing reliable guidance for metabolic engineering.


Assuntos
Simulação por Computador , Proteínas de Escherichia coli , Escherichia coli , Redes e Vias Metabólicas , Modelos Biológicos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Engenharia Metabólica , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
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