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1.
Proc Natl Acad Sci U S A ; 119(11): e2117013119, 2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35259022

RESUMO

SignificanceThe study provided a long-sought molecular mechanism that could explain the link between fatty acid metabolism and cancer metastasis. Further understanding may lead to new strategies to inhibit cancer metastasis. The chemical proteomic approach developed here will be useful for discovering other regulatory mechanisms of protein function by small molecule metabolites.


Assuntos
Acil Coenzima A/metabolismo , Nucleosídeo NM23 Difosfato Quinases/antagonistas & inibidores , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias da Mama , Endocitose , Feminino , Humanos , Metástase Neoplásica , Neoplasias/etiologia , Ligação Proteica , Proteoma , Proteômica/métodos
2.
J Biol Chem ; 298(4): 101735, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35181339

RESUMO

Dihydroceramide is a lipid molecule generated via the action of (dihydro)ceramide synthases (CerSs), which use two substrates, namely sphinganine and fatty acyl-CoAs. Sphinganine is generated via the sequential activity of two integral membrane proteins located in the endoplasmic reticulum. Less is known about the source of the fatty acyl-CoAs, although a number of cytosolic proteins in the pathways of acyl-CoA generation modulate ceramide synthesis via direct or indirect interaction with the CerSs. In this study, we demonstrate, by proteomic analysis of immunoprecipitated proteins, that fatty acid transporter protein 2 (FATP2) (also known as very long-chain acyl-CoA synthetase) directly interacts with CerS2 in mouse liver. Studies in cultured cells demonstrated that other members of the FATP family can also interact with CerS2, with the interaction dependent on both proteins being catalytically active. In addition, transfection of cells with FATP1, FATP2, or FATP4 increased ceramide levels although only FATP2 and 4 increased dihydroceramide levels, consistent with their known intracellular locations. Finally, we show that lipofermata, an FATP2 inhibitor which is believed to directly impact tumor cell growth via modulation of FATP2, decreased de novo dihydroceramide synthesis, suggesting that some of the proposed therapeutic effects of lipofermata may be mediated via (dihydro)ceramide rather than directly via acyl-CoA generation. In summary, our study reinforces the idea that manipulating the pathway of fatty acyl-CoA generation will impact a wide variety of down-stream lipids, not least the sphingolipids, which utilize two acyl-CoA moieties in the initial steps of their synthesis.


Assuntos
Ceramidas , Coenzima A Ligases , Esfingosina N-Aciltransferase , Acil Coenzima A/metabolismo , Animais , Ceramidas/biossíntese , Fígado/metabolismo , Camundongos , Oxirredutases/metabolismo , Proteômica , Esfingosina N-Aciltransferase/metabolismo
3.
Proc Natl Acad Sci U S A ; 119(7)2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35140179

RESUMO

S-acylation, also known as palmitoylation, is the most abundant form of protein lipidation in humans. This reversible posttranslational modification, which targets thousands of proteins, is catalyzed by 23 members of the DHHC family of integral membrane enzymes. DHHC enzymes use fatty acyl-CoA as the ubiquitous fatty acyl donor and become autoacylated at a catalytic cysteine; this intermediate subsequently transfers the fatty acyl group to a cysteine in the target protein. Protein S-acylation intersects with almost all areas of human physiology, and several DHHC enzymes are considered as possible therapeutic targets against diseases such as cancer. These efforts would greatly benefit from a detailed understanding of the molecular basis for this crucial enzymatic reaction. Here, we combine X-ray crystallography with all-atom molecular dynamics simulations to elucidate the structure of the precatalytic complex of human DHHC20 in complex with palmitoyl CoA. The resulting structure reveals that the fatty acyl chain inserts into a hydrophobic pocket within the transmembrane spanning region of the protein, whereas the CoA headgroup is recognized by the cytosolic domain through polar and ionic interactions. Biochemical experiments corroborate the predictions from our structural model. We show, using both computational and experimental analyses, that palmitoyl CoA acts as a bivalent ligand where the interaction of the DHHC enzyme with both the fatty acyl chain and the CoA headgroup is important for catalytic chemistry to proceed. This bivalency explains how, in the presence of high concentrations of free CoA under physiological conditions, DHHC enzymes can efficiently use palmitoyl CoA as a substrate for autoacylation.


Assuntos
Acil Coenzima A/química , Acil Coenzima A/metabolismo , Aciltransferases/metabolismo , Aciltransferases/genética , Domínio Catalítico , Membrana Celular/enzimologia , Regulação Enzimológica da Expressão Gênica , Humanos , Lipoilação , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Conformação Proteica , Domínios Proteicos
4.
Proc Natl Acad Sci U S A ; 119(8)2022 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-35165190

RESUMO

Mycobacterium tuberculosis has a lipid-rich cell envelope that is remodeled throughout infection to enable adaptation within the host. Few transcriptional regulators have been characterized that coordinate synthesis of mycolic acids, the major cell wall lipids of mycobacteria. Here, we show that the mycolic acid desaturase regulator (MadR), a transcriptional repressor of the mycolate desaturase genes desA1 and desA2, controls mycolic acid desaturation and biosynthesis in response to cell envelope stress. A madR-null mutant of M. smegmatis exhibited traits of an impaired cell wall with an altered outer mycomembrane, accumulation of a desaturated α-mycolate, susceptibility to antimycobacterials, and cell surface disruption. Transcriptomic profiling showed that enriched lipid metabolism genes that were significantly down-regulated upon madR deletion included acyl-coenzyme A (aceyl-CoA) dehydrogenases, implicating it in the indirect control of ß-oxidation pathways. Electromobility shift assays and binding affinities suggest a unique acyl-CoA pool-sensing mechanism, whereby MadR is able to bind a range of acyl-CoAs, including those with unsaturated as well as saturated acyl chains. MadR repression of desA1/desA2 is relieved upon binding of saturated acyl-CoAs of chain length C16 to C24, while no impact is observed upon binding of shorter chain and unsaturated acyl-CoAs. We propose this mechanism of regulation as distinct to other mycolic acid and fatty acid synthesis regulators and place MadR as the key regulatory checkpoint that coordinates mycolic acid remodeling during infection in response to host-derived cell surface perturbation.


Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium/metabolismo , Ácidos Micólicos/metabolismo , Racemases e Epimerases/metabolismo , Acil Coenzima A/metabolismo , Proteínas de Bactérias/fisiologia , Parede Celular/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos/metabolismo , Metabolismo dos Lipídeos/fisiologia , Infecções por Mycobacterium , Mycobacterium tuberculosis/metabolismo , Racemases e Epimerases/fisiologia , Fatores de Transcrição/metabolismo
5.
Cell ; 185(3): 513-529.e21, 2022 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-35120663

RESUMO

The human gut microbiota resides within a diverse chemical environment challenging our ability to understand the forces shaping this ecosystem. Here, we reveal that fitness of the Bacteroidales, the dominant order of bacteria in the human gut, is an emergent property of glycans and one specific metabolite, butyrate. Distinct sugars serve as strain-variable fitness switches activating context-dependent inhibitory functions of butyrate. Differential fitness effects of butyrate within the Bacteroides are mediated by species-level variation in Acyl-CoA thioesterase activity and nucleotide polymorphisms regulating an Acyl-CoA transferase. Using in vivo multi-omic profiles, we demonstrate Bacteroides fitness in the human gut is associated together, but not independently, with Acyl-CoA transferase expression and butyrate. Our data reveal that each strain of the Bacteroides exists within a unique fitness landscape based on the interaction of chemical components unpredictable by the effect of each part alone mediated by flexibility in the core genome.


Assuntos
Microbioma Gastrointestinal , Metaboloma , Polissacarídeos/metabolismo , Acil Coenzima A/metabolismo , Sequência de Aminoácidos , Aminoácidos de Cadeia Ramificada/metabolismo , Bacteroidetes/efeitos dos fármacos , Bacteroidetes/genética , Bacteroidetes/crescimento & desenvolvimento , Butiratos/química , Butiratos/farmacologia , Coenzima A-Transferases/química , Coenzima A-Transferases/metabolismo , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/genética , Variação Genética/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Metaboloma/efeitos dos fármacos , Metaboloma/genética , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Especificidade da Espécie , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Transcrição Genética/efeitos dos fármacos
6.
Anal Chem ; 94(6): 2732-2739, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35119811

RESUMO

Acyl-coenzyme A derivatives (acyl-CoAs) are core molecules in the fatty acid and energy metabolism across all species. However, in vivo, many other carboxylic acids can form xenobiotic acyl-CoA esters, including drugs. More than 2467 acyl-CoAs are known from the published literature. In addition, more than 300 acyl-CoAs are covered in pathway databases, but as of October 2020, only 53 experimental acyl-CoA tandem mass spectra are present in NIST20 and MoNA libraries to enable annotation of the mass spectra in untargeted metabolomics studies. The experimental spectra originated from low-resolution ion trap and triple quadrupole mass spectrometers as well as high-resolution quadrupole-time of flight and orbital ion trap instruments at various collision energies. We used MassFrontier software and the literature to annotate fragment ions to generate fragmentation rules and intensities for the different instruments and collision energies. These rules were then applied to 1562 unique species based on [M+H]+ and [M-H]- precursor ions to generate two mass spectra per instrument platform and collision energy, amassing an in silico library of 10,934 accurate mass MS/MS spectra that are freely available at github.com/urikeshet/CoA-Blast. The spectra can be imported into a commercial or freely available mass spectral search tool. We used the libraries to annotate 23 acyl-CoA esters in mouse liver, including 8 novel species.


Assuntos
Acil Coenzima A , Espectrometria de Massas em Tandem , Acil Coenzima A/metabolismo , Animais , Fígado/metabolismo , Metabolômica , Camundongos , Software
7.
Int J Med Sci ; 19(1): 89-97, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34975302

RESUMO

Ageing is one of the major risk factors of human diseases, including cancer, diabetes, and cardiovascular disease. Mulberry exhibits a wide range of functions, such as anti-oxidant, anti-inflammation, and anti-diabetes. In this study, we investigated the role of mulberry polyphenol extract (MPE) in K-Ras-induced senescence of smooth muscle cells. Forced expression of K-Ras enhanced senescence of smooth muscle A7r5 cells as shown by the elevation of ß-galactosidase activity. Treatment with MPE significantly repressed the Ras, phosphorylated ERK, and ß-galactosidase level. MPE triggered the association of cyclins with their corresponding cyclin-dependent protein kinases and hyperphosphorylated retinoblastoma (RB). MPE also down-regulated the levels of K-Ras-induced CDK inhibitors. MPE enhanced the phosphorylated AMP-dependent protein kinase (AMPK) and inducible nitric oxide synthase (iNOS) level in the presence of K-Ras. Pretreatment with either L-NAME or AMPK inhibitor reversed the effects of MPE. In addition, L-NAME and AMPK inhibitor repressed the MPE-induced total and phosphorylated 3-hydroxy-3-methylglutaryl coenzyme A (HMG-Co A) level. MPE repressed K-Ras-induced G0/G1 arrest, whereas L-NAME and AMPK inhibitor blocked the effects of MPE. Our results indicated that MPE recovered the K-Ras-induced senescence of vascular smooth muscle cells through iNOS and AMPK-dependent pathway. Our findings suggested that MPE may prevent ageing-induced atherosclerosis.


Assuntos
Senescência Celular/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Morus/química , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Extratos Vegetais/farmacologia , Polifenóis/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Acil Coenzima A/metabolismo , Células Cultivadas , Expressão Gênica , Humanos , Miócitos de Músculo Liso/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Fosforilação , Proteólise , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , beta-Galactosidase/metabolismo
8.
Plant Sci ; 315: 111130, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35067300

RESUMO

Soil salinization is a major factor impacting modern agricultural production, and alkaline soils contain large amounts of NaHCO3. Therefore, understanding plant tolerance to high levels of NaHCO3 is essential. In this study, a transcriptome analysis of shoot and root tissues of wild-type Arabidopsis thaliana was conducted at 0, 4, 12, 24 and 48 h after exposure to a 3 mM NaHCO3 stress. We focused on differentially expressed genes (DEGs) in roots identified in the early stages (4 h and 12 h) of the NaHCO3 stress response that were enriched in GO term, carboxylic acid metabolic process, and utilize HCO3-. Six genes were identified that exhibited similar expression patterns in both the RNA-seq and qRT-PCR data. We also characterized the phenotypic response of AtMCCA-overexpressing plants to carbonate stress, and found that the ability of AtMCCA-overexpressing plants to tolerate carbonate stress was enhanced by the addition of biotin to the growth medium.


Assuntos
Acil Coenzima A/genética , Acil Coenzima A/metabolismo , Adaptação Fisiológica/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Carbamatos/efeitos adversos , Transcriptoma , Regulação da Expressão Gênica de Plantas , Genes de Plantas
9.
Xenobiotica ; 52(1): 16-25, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35084285

RESUMO

Some drugs with carboxylic acid moieties can potentially cause rare but severe hepatotoxicity. The reactive chemical species generated by drug metabolism are thought to be one reason for this event. Although the phase II conjugation metabolism of carboxylic acids generally renders a compound more polar and inactive, it is also responsible for the formation of reactive metabolites.This study aimed to provide a new approach towards the risk assessment of carboxylic acids in the aspect of reactive acyl CoA metabolites.Although acyl CoA metabolites have been concerned, it is difficult to detect them because of their instability. We investigated the trapping agents for acyl CoA metabolites. We found that cysteine is a good trapping agent and developed an assay method for the reactivity of acyl CoA metabolites. We evaluated 17 drugs with carboxylic acid moieties, all drugs concerned with hepatotoxicity displayed reactive potential. With consideration of the exposure of each parent drug, the correlation between drug labels and the calculated risk of carboxylic drugs was improved.These evaluations can be conducted without radiochemical reagents or the authentic standards of metabolites. We believe that the method will be beneficial for drug discovery.


Assuntos
Acil Coenzima A , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Acil Coenzima A/química , Acil Coenzima A/metabolismo , Ácidos Carboxílicos/metabolismo , Cisteína , Humanos , Medição de Risco
10.
Cells ; 11(2)2022 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-35053322

RESUMO

Skeletal muscle is perceived as a major tissue in glucose and lipid metabolism. High fat diet (HFD) lead to the accumulation of intramuscular lipids, including: long chain acyl-CoA, diacylglycerols, and ceramides. Ceramides are considered to be one of the most important lipid groups in the generation of skeletal muscle insulin resistance. So far, it has not been clearly established whether all ceramides adversely affect the functioning of the insulin pathway, or whether there are certain ceramide species that play a pivotal role in the induction of insulin resistance. Therefore, we designed a study in which the expression of CerS1 and CerS5 genes responsible for the synthesis of C18:0-Cer and C16:0-Cer, respectively, was locally silenced in the gastrocnemius muscle of HFD-fed mice through in vivo electroporation-mediated shRNA plasmids. Our study indicates that HFD feeding induced both, the systemic and skeletal muscle insulin resistance, which was accompanied by an increase in the intramuscular lipid levels, decreased activation of the insulin pathway and, consequently, a decrease in the skeletal muscle glucose uptake. CerS1 silencing leads to a reduction in C18:0-Cer content, with a subsequent increase in the activity of the insulin pathway, and an improvement in skeletal muscle glucose uptake. Such effects were not visible in case of CerS5 silencing, which indicates that the accumulation of C18:0-Cer plays a decisive role in the induction of skeletal muscle insulin resistance.


Assuntos
Inativação Gênica , Glucose/metabolismo , Resistência à Insulina/genética , Proteínas de Membrana/genética , Músculo Esquelético/metabolismo , Esfingosina N-Aciltransferase/genética , Acil Coenzima A/metabolismo , Animais , Dieta Hiperlipídica , Diglicerídeos/metabolismo , Ácidos Graxos/sangue , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Insulina/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Transdução de Sinais , Esfingolipídeos/metabolismo , Esfingosina N-Aciltransferase/metabolismo
11.
Metab Eng ; 69: 262-274, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34883244

RESUMO

Short-chain esters have broad utility as flavors, fragrances, solvents, and biofuels. Controlling selectivity of ester microbial biosynthesis has been an outstanding metabolic engineering problem. In this study, we enabled the de novo fermentative microbial biosynthesis of butyryl-CoA-derived designer esters (e.g., butyl acetate, ethyl butyrate, butyl butyrate) in Escherichia coli with controllable selectivity. Using the modular design principles, we generated the butyryl-CoA-derived ester pathways as exchangeable production modules compatible with an engineered chassis cell for anaerobic production of designer esters. We designed these modules derived from an acyl-CoA submodule (e.g., acetyl-CoA, butyryl-CoA), an alcohol submodule (e.g., ethanol, butanol), a cofactor regeneration submodule (e.g., NADH), and an alcohol acetyltransferase (AAT) submodule (e.g., ATF1, SAAT) for rapid module construction and optimization by manipulating replication (e.g., plasmid copy number), transcription (e.g., promoters), translation (e.g., codon optimization), pathway enzymes, and pathway induction conditions. To further enhance production of designer esters with high selectivity, we systematically screened various strategies of protein solubilization using protein fusion tags and chaperones to improve the soluble expression of multiple pathway enzymes. Finally, our engineered ester-producing strains could achieve 19-fold increase in butyl acetate production (0.64 g/L, 96% selectivity), 6-fold increase in ethyl butyrate production (0.41 g/L, 86% selectivity), and 13-fold increase in butyl butyrate production (0.45 g/L, 54% selectivity) as compared to the initial strains. Overall, this study presented a generalizable framework to engineer modular microbial platforms for anaerobic production of butyryl-CoA-derived designer esters from renewable feedstocks.


Assuntos
Ésteres , Engenharia Metabólica , Acil Coenzima A/genética , Acil Coenzima A/metabolismo , Ésteres/metabolismo , Etanol/metabolismo
12.
Mol Cell ; 82(2): 447-462.e6, 2022 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-34856123

RESUMO

Quantitative subcellular metabolomic measurements can explain the roles of metabolites in cellular processes but are subject to multiple confounding factors. We developed stable isotope labeling of essential nutrients in cell culture-subcellular fractionation (SILEC-SF), which uses isotope-labeled internal standard controls that are present throughout fractionation and processing to quantify acyl-coenzyme A (acyl-CoA) thioesters in subcellular compartments by liquid chromatography-mass spectrometry. We tested SILEC-SF in a range of sample types and examined the compartmentalized responses to oxygen tension, cellular differentiation, and nutrient availability. Application of SILEC-SF to the challenging analysis of the nuclear compartment revealed a nuclear acyl-CoA profile distinct from that of the cytosol, with notable nuclear enrichment of propionyl-CoA. Using isotope tracing, we identified the branched chain amino acid isoleucine as a major metabolic source of nuclear propionyl-CoA and histone propionylation, thus revealing a new mechanism of crosstalk between metabolism and the epigenome.


Assuntos
Acil Coenzima A/metabolismo , Compartimento Celular , Núcleo Celular/metabolismo , Metabolismo Energético , Histonas/metabolismo , Metabolômica , Processamento de Proteína Pós-Traducional , Animais , Diferenciação Celular , Cromatografia Líquida , Citosol/metabolismo , Epigênese Genética , Células Hep G2 , Humanos , Isoleucina , Metaboloma , Camundongos , Mitocôndrias/metabolismo , Oxigênio/metabolismo , Espectrometria de Massas por Ionização por Electrospray
13.
J Biol Chem ; 298(2): 101517, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34942147

RESUMO

Ceramide is a lipid moiety synthesized via the enzymatic activity of ceramide synthases (CerSs), six of which have been identified in mammalian cells, and each of which uses a unique subset of acyl-CoAs for ceramide synthesis. The CerSs are part of a larger gene family, the Tram-Lag-CLN8 domain family. Here, we identify a unique, C-terminal motif, the DxRSDxE motif, which is only found in CerSs and not in other Tram-Lag-CLN8 family members. Deletion of this motif in either CerS2 or in CerS6 did not affect the ability of either enzyme to generate ceramide using both an in vitro assay and metabolic labeling, but deletion of this motif did affect the activity of CerS2 when coexpressed with CerS6. Surprisingly, transfection of cells with either CerS2 or CerS6 lacking the motif did not result in changes in cellular ceramide levels. We found that CerS2 and CerS6 interact with each other, as shown by immunoprecipitation, but deletion of the DxRSDxE motif impeded this interaction. Moreover, proteomics analysis of cells transfected with CerS6Δ338-344 indicated that deletion of the C-terminal motif impacted cellular protein expression, and in particular, the levels of ORMDL1, a negative regulator of sphingolipid synthesis. We suggest that this novel C-terminal motif regulates CerS dimer formation and thereby impacts ceramide synthesis.


Assuntos
Acil Coenzima A , Ceramidas , Acil Coenzima A/metabolismo , Animais , Ceramidas/metabolismo , Mamíferos/metabolismo , Proteômica , Esfingosina N-Aciltransferase/genética , Esfingosina N-Aciltransferase/metabolismo
14.
J Biol Chem ; 298(1): 101522, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34952003

RESUMO

Actinobacterial 2-hydroxyacyl-CoA lyase reversibly catalyzes the thiamine diphosphate-dependent cleavage of 2-hydroxyisobutyryl-CoA to formyl-CoA and acetone. This enzyme has great potential for use in synthetic one-carbon assimilation pathways for sustainable production of chemicals, but lacks details of substrate binding and reaction mechanism for biochemical reengineering. We determined crystal structures of the tetrameric enzyme in the closed conformation with bound substrate, covalent postcleavage intermediate, and products, shedding light on active site architecture and substrate interactions. Together with molecular dynamics simulations of the covalent precleavage complex, the complete catalytic cycle is structurally portrayed, revealing a proton transfer from the substrate acyl Cß hydroxyl to residue E493 that returns it subsequently to the postcleavage Cα-carbanion intermediate. Kinetic parameters obtained for mutants E493A, E493Q, and E493K confirm the catalytic role of E493 in the WT enzyme. However, the 10- and 50-fold reduction in lyase activity in the E493A and E493Q mutants, respectively, compared with WT suggests that water molecules may contribute to proton transfer. The putative catalytic glutamate is located on a short α-helix close to the active site. This structural feature appears to be conserved in related lyases, such as human 2-hydroxyacyl-CoA lyase 2. Interestingly, a unique feature of the actinobacterial 2-hydroxyacyl-CoA lyase is a large C-terminal lid domain that, together with active site residues L127 and I492, restricts substrate size to ≤C5 2-hydroxyacyl residues. These details about the catalytic mechanism and determinants of substrate specificity pave the ground for designing tailored catalysts for acyloin condensations for one-carbon and short-chain substrates in biotechnological applications.


Assuntos
Liases , Acil Coenzima A/metabolismo , Carbono , Catálise , Domínio Catalítico , Cristalografia por Raios X , Humanos , Liases/metabolismo , Prótons , Especificidade por Substrato
15.
J Biol Chem ; 298(1): 101474, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34896395

RESUMO

Mycofactocin (MFT) is a ribosomally synthesized and post-translationally-modified redox cofactor found in pathogenic mycobacteria. While MFT biosynthetic proteins have been extensively characterized, the physiological conditions under which MFT biosynthesis is required are not well understood. To gain insights into the mechanisms of regulation of MFT expression in Mycobacterium smegmatis mc2155, we investigated the DNA-binding and ligand-binding activities of the putative TetR-like transcription regulator, MftR. In this study, we demonstrated that MftR binds to the mft promoter region. We used DNase I footprinting to identify the 27 bp palindromic operator located 5' to mftA and found it to be highly conserved in Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium ulcerans, and Mycobacterium marinum. To determine under which conditions the mft biosynthetic gene cluster (BGC) is induced, we screened for effectors of MftR. As a result, we found that MftR binds to long-chain acyl-CoAs with low micromolar affinities. To demonstrate that oleoyl-CoA induces the mft BGC in vivo, we re-engineered a fluorescent protein reporter system to express an MftA-mCherry fusion protein. Using this mCherry fluorescent readout, we show that the mft BGC is upregulated in M. smegmatis mc2155 when oleic acid is supplemented to the media. These results suggest that MftR controls expression of the mft BGC and that MFT production is induced by long-chain acyl-CoAs. Since MFT-dependent dehydrogenases are known to colocalize with acyl carrier protein/CoA-modifying enzymes, these results suggest that MFT might be critical for fatty acid metabolism or cell wall reorganization.


Assuntos
Acil Coenzima A , Proteínas de Bactérias , Mycobacterium , Acil Coenzima A/genética , Acil Coenzima A/metabolismo , Proteínas de Bactérias/metabolismo , Mycobacterium/enzimologia , Mycobacterium/metabolismo , Mycobacterium marinum/metabolismo , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/metabolismo , Oxirredução
16.
Gene ; 809: 146010, 2022 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-34688814

RESUMO

Synthetic biology requires well-characterized biological parts that can be combined into functional modules. One type of biological parts are transcriptional regulators and their cognate operator elements, which enable to either generate an input-specific response or are used as actuator modules. A range of regulators has already been characterized and used for orthogonal gene expression engineering, however, previous efforts have mostly focused on bacterial regulators. This work aims to design and explore the use of an archaeal TetR family regulator, FadRSa from Sulfolobus acidocaldarius, in a bacterial system, namely Escherichia coli. This is a challenging objective given the fundamental difference between the bacterial and archaeal transcription machinery and the lack of a native TetR-like FadR regulatory system in E. coli. The synthetic σ70-dependent bacterial promoter proD was used as a starting point to design hybrid bacterial/archaeal promoter/operator regions, in combination with the mKate2 fluorescent reporter enabling a readout. Four variations of proD containing FadRSa binding sites were constructed and characterized. While expressional activity of the modified promoter proD was found to be severely diminished for two of the constructs, constructs in which the binding site was introduced adjacent to the -35 promoter element still displayed sufficient basal transcriptional activity and showed up to 7-fold repression upon expression of FadRSa. Addition of acyl-CoA has been shown to disrupt FadRSa binding to the DNA in vitro. However, extracellular concentrations of up to 2 mM dodecanoate, subsequently converted to acyl-CoA by the cell, did not have a significant effect on repression in the bacterial system. This work demonstrates that archaeal transcription regulators can be used to generate actuator elements for use in E. coli, although the lack of ligand response underscores the challenge of maintaining biological function when transferring parts to a phylogenetically divergent host.


Assuntos
Proteínas Arqueais/genética , Escherichia coli/genética , Engenharia Genética/métodos , Fatores de Transcrição/genética , Acil Coenzima A/genética , Acil Coenzima A/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Ácidos Graxos/metabolismo , Regulação Bacteriana da Expressão Gênica , Isopropiltiogalactosídeo/farmacologia , Lauratos/farmacologia , Microrganismos Geneticamente Modificados , Regiões Operadoras Genéticas , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Sulfolobus acidocaldarius/genética
17.
Sci Rep ; 11(1): 22849, 2021 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-34819551

RESUMO

The ammonia-oxidizing thaumarchaeal 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle is one of the most energy-efficient CO2 fixation cycles discovered thus far. The protein encoded by Nmar_1308 (from Nitrosopumilus maritimus SCM1) is a promiscuous enzyme that catalyzes two essential reactions within the thaumarchaeal 3HP/4HB cycle, functioning as both a crotonyl-CoA hydratase (CCAH) and 3-hydroxypropionyl-CoA dehydratase (3HPD). In performing both hydratase and dehydratase activities, Nmar_1308 reduces the total number of enzymes necessary for CO2 fixation in Thaumarchaeota, reducing the overall cost for biosynthesis. Here, we present the first high-resolution crystal structure of this bifunctional enzyme with key catalytic residues in the thaumarchaeal 3HP/4HB pathway.


Assuntos
Acil Coenzima A/metabolismo , Archaea/enzimologia , Proteínas Arqueais/metabolismo , Dióxido de Carbono/metabolismo , Enoil-CoA Hidratase/metabolismo , Archaea/genética , Proteínas Arqueais/química , Proteínas Arqueais/genética , Catálise , Cristalografia por Raios X , Enoil-CoA Hidratase/química , Enoil-CoA Hidratase/genética , Modelos Moleculares , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato
18.
Nat Commun ; 12(1): 6869, 2021 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-34824256

RESUMO

As the major component of cell membranes, phosphatidylcholine (PC) is synthesized de novo in the Kennedy pathway and then undergoes extensive deacylation-reacylation remodeling via Lands' cycle. The re-acylation is catalyzed by lysophosphatidylcholine acyltransferase (LPCAT) and among the four LPCAT members in human, the LPCAT3 preferentially introduces polyunsaturated acyl onto the sn-2 position of lysophosphatidylcholine, thereby modulating the membrane fluidity and membrane protein functions therein. Combining the x-ray crystallography and the cryo-electron microscopy, we determined the structures of LPCAT3 in apo-, acyl donor-bound, and acyl receptor-bound states. A reaction chamber was revealed in the LPCAT3 structure where the lysophosphatidylcholine and arachidonoyl-CoA were positioned in two tunnels connected near to the catalytic center. A side pocket was found expanding the tunnel for the arachidonoyl CoA and holding the main body of arachidonoyl. The structural and functional analysis provides the basis for the re-acylation of lysophosphatidylcholine and the substrate preference during the reactions.


Assuntos
1-Acilglicerofosfocolina O-Aciltransferase/química , Fosfolipídeos/química , 1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Acil Coenzima A/química , Acil Coenzima A/metabolismo , Acilação , Animais , Domínio Catalítico , Galinhas , Microscopia Crioeletrônica , Cristalografia por Raios X , Lisofosfatidilcolinas/química , Lisofosfatidilcolinas/metabolismo , Modelos Moleculares , Fosfolipídeos/metabolismo , Multimerização Proteica , Relação Estrutura-Atividade , Especificidade por Substrato
19.
PLoS One ; 16(10): e0257478, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34618820

RESUMO

BACKGROUND: Montbretins are rare specialized metabolites found in montbretia (Crocosmia x crocosmiiflora) corms. Montbretin A (MbA) is of particular interest as a novel therapeutic for type-2 diabetes and obesity. There is no scalable production system for this complex acylated flavonol glycoside. MbA biosynthesis has been reconstructed in Nicotiana benthamiana using montbretia genes for the assembly of MbA from its various different building blocks. However, in addition to smaller amounts of MbA, the therapeutically inactive montbretin B (MbB) was the major product of this metabolic engineering effort. MbA and MbB differ in a single hydroxyl group of their acyl side chains, which are derived from caffeoyl-CoA and coumaroyl-CoA, respectively. Biosynthesis of both MbA and MbB also require coumaroyl-CoA for the formation of the myricetin core. Caffeoyl-CoA and coumaroyl-CoA are formed in the central phenylpropanoid pathway by acyl activating enzymes (AAEs) known as 4-coumaroyl-CoA ligases (4CLs). Here we investigated a small family of montbretia AAEs and 4CLs, and their possible contribution to montbretin biosynthesis. RESULTS: Transcriptome analysis for gene expression patterns related to montbretin biosynthesis identified eight different montbretia AAEs belonging to four different clades. Enzyme characterization identified 4CL activity for two clade IV members, Cc4CL1 and Cc4CL2, converting different hydroxycinnamic acids into the corresponding CoA thioesters. Both enzymes preferred coumaric acid over caffeic acid as a substrate in vitro. While expression of montbretia AAEs did not enhance MbA biosynthesis in N. benthamiana, we demonstrated that both Cc4CLs can be used to activate coumaric and caffeic acid towards flavanone biosynthesis in yeast (Saccharomyces cerevisiae). CONCLUSIONS: Montbretia expresses two functional 4CLs, but neither of them is specific for the formation of caffeoyl-CoA. Based on differential expression analysis and phylogeny Cc4CL1 is most likely involved in MbA biosynthesis, while Cc4CL2 may contribute to lignin biosynthesis. Both Cc4CLs can be used for flavanone production to support metabolic engineering of MbA in yeast.


Assuntos
Acil Coenzima A/metabolismo , Flavonas/metabolismo , Hipoglicemiantes/metabolismo , Iridaceae/metabolismo , Ligases/metabolismo , Proteínas de Plantas/metabolismo , Trissacarídeos/metabolismo , Acil Coenzima A/genética , Vias Biossintéticas , Flavonas/genética , Regulação da Expressão Gênica de Plantas , Engenharia Genética , Iridaceae/genética , Ligases/genética , Engenharia Metabólica , Proteínas de Plantas/genética , Tabaco/genética , Tabaco/metabolismo , Trissacarídeos/genética
20.
Sci Rep ; 11(1): 20842, 2021 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-34675283

RESUMO

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is a crucial enzyme in the ergosterol biosynthesis pathway. The aim of this study was to obtain, purify, characterize, and overexpress five point mutations in highly conserved regions of the catalytic domain of Candida glabrata HMGR (CgHMGR) to explore the function of key amino acid residues in enzymatic activity. Glutamic acid (Glu) was substituted by glutamine in the E680Q mutant (at the dimerization site), Glu by glutamine in E711Q (at the substrate binding site), aspartic acid by alanine in D805A, and methionine by arginine in M807R (the latter two at the cofactor binding site). A double mutation, E680Q-M807R, was included. Regarding recombinant and wild-type CgHMGR, in vitro enzymatic activity was significantly lower for the former, as was the in silico binding energy of simvastatin, alpha-asarone and the HMG-CoA substrate. E711Q displayed the lowest enzymatic activity and binding energy, suggesting the importance of Glu711 (in the substrate binding site). The double mutant CgHMGR E680Q-M807R exhibited the second lowest enzymatic activity. Based on the values of the kinetic parameters KM and Vmax, the mutated amino acids appear to participate in binding. The current findings provide insights into the role of residues in the catalytic site of CgHMGR.


Assuntos
Acil Coenzima A/genética , Candida glabrata/genética , Proteínas Fúngicas/genética , Mutação Puntual , Acil Coenzima A/química , Acil Coenzima A/metabolismo , Sítios de Ligação , Candida glabrata/química , Candida glabrata/metabolismo , Domínio Catalítico , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Modelos Moleculares , Filogenia , Especificidade por Substrato
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