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1.
Nutrients ; 16(11)2024 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-38892698

RESUMO

One-carbon metabolism (OCM) is a complex and interconnected network that undergoes drastic changes during pregnancy. In this study, we investigated the longitudinal distribution of OCM-related metabolites in maternal and cord blood and explored their relationships. Additionally, we conducted cross-sectional analyses to examine the interrelationships among these metabolites. This study included 146 healthy pregnant women who participated in the Chiba Study of Mother and Child Health. Maternal blood samples were collected during early pregnancy, late pregnancy, and delivery, along with cord blood samples. We analyzed 18 OCM-related metabolites in serum using stable isotope dilution liquid chromatography/tandem mass spectrometry. We found that serum S-adenosylmethionine (SAM) concentrations in maternal blood remained stable throughout pregnancy. Conversely, S-adenosylhomocysteine (SAH) concentrations increased, and the total homocysteine/total cysteine ratio significantly increased with advancing gestational age. The betaine/dimethylglycine ratio was negatively correlated with total homocysteine in maternal blood for all sampling periods, and this correlation strengthened with advances in gestational age. Most OCM-related metabolites measured in this study showed significant positive correlations between maternal blood at delivery and cord blood. These findings suggest that maternal OCM status may impact fetal development and indicate the need for comprehensive and longitudinal evaluations of OCM during pregnancy.


Assuntos
Sangue Fetal , Homocisteína , S-Adenosilmetionina , Humanos , Feminino , Sangue Fetal/metabolismo , Sangue Fetal/química , Gravidez , Adulto , Estudos Longitudinais , Homocisteína/sangue , Japão , S-Adenosilmetionina/sangue , S-Adenosil-Homocisteína/sangue , Estudos Transversais , Idade Gestacional , Carbono/metabolismo , Betaína/sangue , Cisteína/sangue , Espectrometria de Massas em Tandem , Glicina/sangue , População do Leste Asiático , Sarcosina/análogos & derivados
2.
Nat Commun ; 15(1): 5167, 2024 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-38886362

RESUMO

Methylenetetrahydrofolate reductase (MTHFR) is a pivotal flavoprotein connecting the folate and methionine methyl cycles, catalyzing the conversion of methylenetetrahydrofolate to methyltetrahydrofolate. Human MTHFR (hMTHFR) undergoes elaborate allosteric regulation involving protein phosphorylation and S-adenosylmethionine (AdoMet)-dependent inhibition, though other factors such as subunit orientation and FAD status remain understudied due to the lack of a functional structural model. Here, we report crystal structures of Chaetomium thermophilum MTHFR (cMTHFR) in both active (R) and inhibited (T) states. We reveal FAD occlusion by Tyr361 in the T-state, which prevents substrate interaction. Remarkably, the inhibited form of cMTHFR accommodates two AdoMet molecules per subunit. In addition, we conducted a detailed investigation of the phosphorylation sites in hMTHFR, three of which were previously unidentified. Based on the structural framework provided by our cMTHFR model, we propose a possible mechanism to explain the allosteric structural transition of MTHFR, including the impact of phosphorylation on AdoMet-dependent inhibition.


Assuntos
Chaetomium , Metilenotetra-Hidrofolato Redutase (NADPH2) , S-Adenosilmetionina , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Metilenotetra-Hidrofolato Redutase (NADPH2)/química , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , S-Adenosilmetionina/metabolismo , S-Adenosilmetionina/química , Regulação Alostérica , Chaetomium/enzimologia , Chaetomium/metabolismo , Chaetomium/genética , Fosforilação , Humanos , Cristalografia por Raios X , Modelos Moleculares , Flavina-Adenina Dinucleotídeo/metabolismo , Flavina-Adenina Dinucleotídeo/química
3.
Nat Commun ; 15(1): 5256, 2024 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-38898040

RESUMO

Archaea possess characteristic membrane-spanning lipids that are thought to contribute to the adaptation to extreme environments. However, the biosynthesis of these lipids is poorly understood. Here, we identify a radical S-adenosyl-L-methionine (SAM) enzyme that synthesizes glycerol monoalkyl glycerol tetraethers (GMGTs). The enzyme, which we name GMGT synthase (Gms), catalyzes the formation of a C(sp3)-C(sp3) linkage between the two isoprenoid chains of glycerol dialkyl glycerol tetraethers (GDGTs). This conclusion is supported by heterologous expression of gene gms from a GMGT-producing species in a methanogen, as well as demonstration of in vitro activity using purified Gms enzyme. Additionally, we show that genes encoding putative Gms homologs are present in obligate anaerobic archaea and in metagenomes obtained from oxygen-deficient environments, and appear to be absent in metagenomes from oxic settings.


Assuntos
Archaea , Oxigênio , S-Adenosilmetionina , S-Adenosilmetionina/metabolismo , Archaea/genética , Archaea/metabolismo , Archaea/enzimologia , Oxigênio/metabolismo , Anaerobiose , Proteínas Arqueais/metabolismo , Proteínas Arqueais/genética , Glicerol/metabolismo , Metagenoma , Filogenia
4.
J Am Chem Soc ; 146(27): 18722-18729, 2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-38943667

RESUMO

Methylation, a widely occurring natural modification serving diverse regulatory and structural functions, is carried out by a myriad of S-adenosyl-l-methionine (AdoMet)-dependent methyltransferases (MTases). The AdoMet cofactor is produced from l-methionine (Met) and ATP by a family of multimeric methionine adenosyltransferases (MAT). To advance mechanistic and functional studies, strategies for repurposing the MAT and MTase reactions to accept extended versions of the transferable group from the corresponding precursors have been exploited. Here, we used structure-guided engineering of mouse MAT2A to enable biocatalytic production of an extended AdoMet analogue, Ado-6-azide, from a synthetic methionine analogue, S-(6-azidohex-2-ynyl)-l-homocysteine (N3-Met). Three engineered MAT2A variants showed catalytic proficiency with the extended analogues and supported DNA derivatization in cascade reactions with M.TaqI and an engineered variant of mouse DNMT1 both in the absence and presence of competing Met. We then installed two of the engineered variants as MAT2A-DNMT1 cascades in mouse embryonic stem cells by using CRISPR-Cas genome editing. The resulting cell lines maintained normal viability and DNA methylation levels and showed Dnmt1-dependent DNA modification with extended azide tags upon exposure to N3-Met in the presence of physiological levels of Met. This for the first time demonstrates a genetically stable system for biosynthetic production of an extended AdoMet analogue, which enables mild metabolic labeling of a DNMT-specific methylome in live mammalian cells.


Assuntos
Metilação de DNA , Metionina Adenosiltransferase , Metionina Adenosiltransferase/metabolismo , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/química , Animais , Camundongos , Engenharia de Proteínas , Epigenoma , S-Adenosilmetionina/metabolismo , S-Adenosilmetionina/química , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , Humanos
5.
SLAS Discov ; 29(4): 100161, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38788976

RESUMO

Methylation of proteins and nucleic acids plays a fundamental role in epigenetic regulation, and discovery of methyltransferase (MT) inhibitors is an area of intense activity. Because of the diversity of MTs and their products, assay methods that detect S-adenosylhomocysteine (SAH) - the invariant product of S-adenosylmethionine (SAM)-dependent methylation reactions - offer some advantages over methods that detect specific methylation events. However, direct, homogenous detection of SAH requires a reagent capable of discriminating between SAH and SAM, which differ by a single methyl group. Moreover, MTs are slow enzymes and many have submicromolar affinities for SAM; these properties translate to a need for detection of SAH at low nanomolar concentrations in the presence of excess SAM. To meet these needs, we leveraged the exquisite molecular recognition properties of a naturally occurring SAH-sensing RNA aptamer, or riboswitch. By splitting the riboswitch into two fragments, such that SAH binding induces assembly of a trimeric complex, we engineered sensors that transduce binding of SAH into positive fluorescence polarization (FP) and time resolved Förster resonance energy transfer (TR-FRET) signals. The split riboswitch configuration, called the AptaFluor™ SAH Methyltransferase Assay, allows robust detection of SAH (Z' > 0.7) at concentrations below 10 nM, with overnight signal stability in the presence of typical MT assay components. The AptaFluor assay tolerates diverse MT substrates, including histones, nucleosomes, DNA and RNA, and we demonstrated its utility as a robust, enzymatic assay method for several methyltransferases with SAM Km values < 1 µM. The assay was validated for HTS by performing a pilot screen of 1,280 compounds against the SARS-CoV-2 RNA capping enzyme, nsp14. By enabling direct, homogenous detection of SAH at low nanomolar concentrations, the AptaFluor assay provides a universal platform for screening and profiling MTs at physiologically relevant SAM concentrations.


Assuntos
Ensaios Enzimáticos , Metiltransferases , Riboswitch , S-Adenosil-Homocisteína , S-Adenosilmetionina , S-Adenosil-Homocisteína/metabolismo , Riboswitch/genética , Metiltransferases/metabolismo , Metiltransferases/genética , Ensaios Enzimáticos/métodos , S-Adenosilmetionina/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Metilação , Humanos , Polarização de Fluorescência/métodos , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética
6.
Mol Biol Cell ; 35(7): ar89, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38696262

RESUMO

Cilia are highly complex motile, sensory, and secretory organelles that contain perhaps 1000 or more distinct protein components, many of which are subject to various posttranslational modifications such as phosphorylation, N-terminal acetylation, and proteolytic processing. Another common modification is the addition of one or more methyl groups to the side chains of arginine and lysine residues. These tunable additions delocalize the side-chain charge, decrease hydrogen bond capacity, and increase both bulk and hydrophobicity. Methylation is usually mediated by S-adenosylmethionine (SAM)-dependent methyltransferases and reversed by demethylases. Previous studies have identified several ciliary proteins that are subject to methylation including axonemal dynein heavy chains that are modified by a cytosolic methyltransferase. Here, we have performed an extensive proteomic analysis of multiple independently derived cilia samples to assess the potential for SAM metabolism and the extent of methylation in these organelles. We find that cilia contain all the enzymes needed for generation of the SAM methyl donor and recycling of the S-adenosylhomocysteine and tetrahydrofolate byproducts. In addition, we find that at least 155 distinct ciliary proteins are methylated, in some cases at multiple sites. These data provide a comprehensive resource for studying the consequences of methyl marks on ciliary biology.


Assuntos
Cílios , Processamento de Proteína Pós-Traducional , Proteômica , S-Adenosilmetionina , Cílios/metabolismo , S-Adenosilmetionina/metabolismo , Metilação , Proteômica/métodos , Animais , Humanos , Metiltransferases/metabolismo , S-Adenosil-Homocisteína/metabolismo , Epigenoma
7.
J Agric Food Chem ; 72(22): 12685-12695, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38771136

RESUMO

Halogenation plays a unique role in the design of agrochemicals. Enzymatic halogenation reactions have attracted great attention due to their excellent specificity and mild reaction conditions. S-adenosyl-l-methionine (SAM)-dependent halogenases mediate the nucleophilic attack of halide ions (X-) to SAM to produce 5'-XDA. However, only 11 SAM-dependent fluorinases and 3 chlorinases have been reported, highlighting the desire for additional halogenases. SAM-dependent hydroxide adenosyltransferase (HATase) has a similar reaction mechanism as halogenases but uses water as a substrate instead of halide ions. Here, we explored a HATase from the thermophile Thermotoga maritima MSB8 and transformed it into a halogenase. We identified a key dyad W8L/V71T for the halogenation reaction. We also obtained the best performing mutants for each halogenation reaction: M1, M2 and M4 for Cl-, Br- and I-, respectively. The M4 mutant retained the thermostability of HATase in the iodination reaction at 80 °C, which surpasses the natural halogenase SalL. QM/MM revealed that these mutants bind halide ions with more suitable angles for nucleophilic attack of C5' of SAM, thus conferring halogenation capabilities. Our work achieved the halide ion specificity of halogenases and generated thermostable halogenases for the first time, which provides new opportunities to expand the halogenase repertoire from hydroxylase.


Assuntos
Proteínas de Bactérias , Thermotoga maritima , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Thermotoga maritima/enzimologia , Thermotoga maritima/genética , Thermotoga maritima/química , Halogenação , Especificidade por Substrato , S-Adenosilmetionina/metabolismo , S-Adenosilmetionina/química , Oxirredutases/química , Oxirredutases/metabolismo , Oxirredutases/genética , Biocatálise
8.
Cell Death Dis ; 15(5): 349, 2024 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-38769167

RESUMO

Osteosarcoma is a malignant bone tumor that primarily inflicts the youth. It often metastasizes to the lungs after chemotherapy failure, which eventually shortens patients' lives. Thus, there is a dire clinical need to develop a novel therapy to tackle osteosarcoma metastasis. Methionine dependence is a special metabolic characteristic of most malignant tumor cells that may offer a target pathway for such therapy. Herein, we demonstrated that methionine deficiency restricted the growth and metastasis of cultured human osteosarcoma cells. A genetically engineered Salmonella, SGN1, capable of overexpressing an L-methioninase and hydrolyzing methionine led to significant reduction of methionine and S-adenosyl-methionine (SAM) specifically in tumor tissues, drastically restricted the growth and metastasis in subcutaneous xenograft, orthotopic, and tail vein-injected metastatic models, and prolonged the survival of the model animals. SGN1 also sharply suppressed the growth of patient-derived organoid and xenograft. Methionine restriction in the osteosarcoma cells initiated severe mitochondrial dysfunction, as evident in the dysregulated gene expression of respiratory chains, increased mitochondrial ROS generation, reduced ATP production, decreased basal and maximum respiration, and damaged mitochondrial membrane potential. Transcriptomic and molecular analysis revealed the reduction of C1orf112 expression as a primary mechanism underlies methionine deprivation-initiated suppression on the growth and metastasis as well as mitochondrial functions. Collectively, our findings unraveled a molecular linkage between methionine restriction, mitochondrial function, and osteosarcoma growth and metastasis. A pharmacological agent, such as SGN1, that can achieve tumor specific deprivation of methionine may represent a promising modality against the metastasis of osteosarcoma and potentially other types of sarcomas as well.


Assuntos
Neoplasias Ósseas , Metionina , Mitocôndrias , Osteossarcoma , Osteossarcoma/patologia , Osteossarcoma/metabolismo , Osteossarcoma/genética , Osteossarcoma/tratamento farmacológico , Metionina/deficiência , Metionina/metabolismo , Humanos , Animais , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Linhagem Celular Tumoral , Camundongos , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Metástase Neoplásica , S-Adenosilmetionina/metabolismo , S-Adenosilmetionina/farmacologia , Camundongos Nus , Espécies Reativas de Oxigênio/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos
9.
Leukemia ; 38(6): 1236-1245, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38643304

RESUMO

Targeting the metabolic dependencies of acute myeloid leukemia (AML) cells is a promising therapeutical strategy. In particular, the cysteine and methionine metabolism pathway (C/M) is significantly altered in AML cells compared to healthy blood cells. Moreover, methionine has been identified as one of the dominant amino acid dependencies of AML cells. Through RNA-seq, we found that the two nucleoside analogs 8-chloro-adenosine (8CA) and 8-amino-adenosine (8AA) significantly suppress the C/M pathway in AML cells, and methionine-adenosyltransferase-2A (MAT2A) is one of most significantly downregulated genes. Additionally, mass spectrometry analysis revealed that Venetoclax (VEN), a BCL-2 inhibitor recently approved by the FDA for AML treatment, significantly decreases the intracellular level of methionine in AML cells. Based on these findings, we hypothesized that combining 8CA or 8AA with VEN can efficiently target the Methionine-MAT2A-S-adenosyl-methionine (SAM) axis in AML. Our results demonstrate that VEN and 8CA/8AA synergistically decrease the SAM biosynthesis and effectively target AML cells both in vivo and in vitro. These findings suggest the promising potential of combining 8CA/8AA and VEN for AML treatment by inhibiting Methionine-MAT2A-SAM axis and provide a strong rationale for our recently activated clinical trial.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes , Sinergismo Farmacológico , Leucemia Mieloide Aguda , Metionina Adenosiltransferase , Metionina , S-Adenosilmetionina , Sulfonamidas , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Humanos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Sulfonamidas/farmacologia , Metionina/metabolismo , Metionina/análogos & derivados , Metionina Adenosiltransferase/metabolismo , Metionina Adenosiltransferase/antagonistas & inibidores , Metionina Adenosiltransferase/genética , Animais , Camundongos , S-Adenosilmetionina/farmacologia , S-Adenosilmetionina/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Linhagem Celular Tumoral
10.
Arch Biochem Biophys ; 756: 110012, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38663796

RESUMO

In recent years, the biological significance of ribosomally synthesized, post-translationally modified peptides (RiPPs) and the intriguing chemistry catalyzed by their tailoring enzymes has garnered significant attention. A subgroup of bacterial radical S-adenosylmethionine (rSAM) enzymes can activate C-H bonds in peptides, which leads to the production of a diverse range of RiPPs. The remarkable ability of these enzymes to facilitate various chemical processes, to generate and harbor high-energy radical species, and to accommodate large substrates with a high degree of flexibility is truly intriguing. The wide substrate scope and diversity of the chemistry performed by rSAM enzymes raise one question: how does the protein environment facilitate these distinct chemical conversions while sharing a similar structural fold? In this review, we discuss recent advances in the field of RiPP-rSAM enzymes, with a particular emphasis on domain architectures and substrate engagements identified by biophysical and structural characterizations. We provide readers with a comparative analysis of six examples of RiPP-rSAM enzymes with experimentally characterized structures. Linking the structural elements and the nature of rSAM-catalyzed RiPP production will provide insight into the functional engineering of enzyme activity to harness their catalytic power in broader applications.


Assuntos
Peptídeos , Processamento de Proteína Pós-Traducional , S-Adenosilmetionina , S-Adenosilmetionina/metabolismo , S-Adenosilmetionina/química , Especificidade por Substrato , Peptídeos/química , Peptídeos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Domínios Proteicos
11.
Nat Commun ; 15(1): 2931, 2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38575566

RESUMO

Cystathionine beta-synthase (CBS) is an essential metabolic enzyme across all domains of life for the production of glutathione, cysteine, and hydrogen sulfide. Appended to the conserved catalytic domain of human CBS is a regulatory domain that modulates activity by S-adenosyl-L-methionine (SAM) and promotes oligomerisation. Here we show using cryo-electron microscopy that full-length human CBS in the basal and SAM-bound activated states polymerises as filaments mediated by a conserved regulatory domain loop. In the basal state, CBS regulatory domains sterically block the catalytic domain active site, resulting in a low-activity filament with three CBS dimers per turn. This steric block is removed when in the activated state, one SAM molecule binds to the regulatory domain, forming a high-activity filament with two CBS dimers per turn. These large conformational changes result in a central filament of SAM-stabilised regulatory domains at the core, decorated with highly flexible catalytic domains. Polymerisation stabilises CBS and reduces thermal denaturation. In PC-3 cells, we observed nutrient-responsive CBS filamentation that disassembles when methionine is depleted and reversed in the presence of SAM. Together our findings extend our understanding of CBS enzyme regulation, and open new avenues for investigating the pathogenic mechanism and therapeutic opportunities for CBS-associated disorders.


Assuntos
Cistationina beta-Sintase , Metionina , Humanos , Cistationina beta-Sintase/metabolismo , Microscopia Crioeletrônica , S-Adenosilmetionina/metabolismo , Domínio Catalítico
12.
Molecules ; 29(8)2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38675528

RESUMO

Glioblastoma (GBM), the most frequent and lethal brain cancer in adults, is characterized by short survival times and high mortality rates. Due to the resistance of GBM cells to conventional therapeutic treatments, scientific interest is focusing on the search for alternative and efficient adjuvant treatments. S-Adenosylmethionine (AdoMet), the well-studied physiological methyl donor, has emerged as a promising anticancer compound and a modulator of multiple cancer-related signaling pathways. We report here for the first time that AdoMet selectively inhibited the viability and proliferation of U87MG, U343MG, and U251MG GBM cells. In these cell lines, AdoMet induced S and G2/M cell cycle arrest and apoptosis and downregulated the expression and activation of proteins involved in homologous recombination DNA repair, including RAD51, BRCA1, and Chk1. Furthermore, AdoMet was able to maintain DNA in a damaged state, as indicated by the increased γH2AX/H2AX ratio. AdoMet promoted mitotic catastrophe through inhibiting Aurora B kinase expression, phosphorylation, and localization causing GBM cells to undergo mitotic catastrophe-induced death. Finally, AdoMet inhibited DNA repair and induced cell cycle arrest, apoptosis, and mitotic catastrophe in patient-derived GBM cells. In light of these results, AdoMet could be considered a potential adjuvant in GBM therapy.


Assuntos
Antineoplásicos , Apoptose , Proliferação de Células , Glioblastoma , S-Adenosilmetionina , Humanos , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patologia , S-Adenosilmetionina/farmacologia , Linhagem Celular Tumoral , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/química , Sobrevivência Celular/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Aurora Quinase B/metabolismo , Aurora Quinase B/antagonistas & inibidores , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Rad51 Recombinase/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Mitose/efeitos dos fármacos
13.
Plant Physiol Biochem ; 210: 108618, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38631157

RESUMO

The Acacia koa S-adenosylmethionine (SAM) synthetase was identified from transcriptome data and cloned into the T7-expression vector pEt14b. Assays indicate a thermoalkaliphic enzyme which tolerates conditions up to pH 10.5, 55 °C and 3 M KCl. In vitro examples of plant SAM-synthetase activity are scarce, however this study provides supporting evidence that these extremophilic properties may actually be typical for this plant enzyme. Enzyme kinetic constants (Km = 1.44 mM, Kcat = 1.29 s-1, Vmax 170 µM. min-1) are comparable to nonplant SAM-synthetases except that substrate inhibition was not apparent at 10 mM ATP/L-methionine. Methods were explored in this study to reduce feedback inhibition, which is known to limit SAM-synthetase activity in vitro. Four single-point mutation variants of the Acacia koa SAM-synthetase were produced, each with varying degrees of reduced reaction rate, greater sensitivity to product inhibition and loss of thermophilic properties. Although an enhanced mutant was not produced, this study describes the first mutagenesis of a plant SAM-synthetase. Overcoming feedback inhibition was accomplished by the addition of organic solvent to enzyme assays. Acetonitrile, methanol or dimethylformamide, when included as 25% of the assay volume, improved total SAM production by 30-65%.


Assuntos
Acacia , Metionina Adenosiltransferase , Acacia/genética , Acacia/metabolismo , Acacia/enzimologia , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Cinética , S-Adenosilmetionina/metabolismo , Concentração de Íons de Hidrogênio
14.
Redox Biol ; 72: 103150, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38599016

RESUMO

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized by impaired motor coordination due to neurological defects and cerebellar dysfunction caused by the accumulation of cholesterol in endolysosomes. Besides the increase in lysosomal cholesterol, mitochondria are also enriched in cholesterol, which leads to decreased membrane fluidity, impaired mitochondrial function and loss of GSH, and has been shown to contribute to the progression of NPC disease. S-Adenosyl-l-methionine (SAM) regulates membrane physical properties through the generation of phosphatidylcholine (PC) from phosphatidylethanolamine (PE) methylation and functions as a GSH precursor by providing cysteine in the transsulfuration pathway. However, the role of SAM in NPC disease has not been investigated. Here we report that Npc1-/- mice exhibit decreased brain SAM levels but unchanged S-adenosyl-l-homocysteine content and lower expression of Mat2a. Brain mitochondria from Npc1-/- mice display decreased mitochondrial GSH levels and liquid chromatography-high resolution mass spectrometry analysis reveal a lower PC/PE ratio in mitochondria, contributing to increased mitochondrial membrane order. In vivo treatment of Npc1-/- mice with SAM restores SAM levels in mitochondria, resulting in increased PC/PE ratio, mitochondrial membrane fluidity and subsequent replenishment of mitochondrial GSH levels. In vivo SAM treatment improves the decline of locomotor activity, increases Purkinje cell survival in the cerebellum and extends the average and maximal life spam of Npc1-/- mice. These findings identify SAM as a potential therapeutic approach for the treatment of NPC disease.


Assuntos
Encéfalo , Glutationa , Fluidez de Membrana , Membranas Mitocondriais , Doença de Niemann-Pick Tipo C , S-Adenosilmetionina , Animais , Camundongos , S-Adenosilmetionina/metabolismo , Membranas Mitocondriais/metabolismo , Doença de Niemann-Pick Tipo C/metabolismo , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Doença de Niemann-Pick Tipo C/genética , Glutationa/metabolismo , Encéfalo/metabolismo , Mitocôndrias/metabolismo , Proteína C1 de Niemann-Pick , Modelos Animais de Doenças , Camundongos Knockout , Fosfatidilcolinas/metabolismo
15.
Nat Commun ; 15(1): 3248, 2024 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-38622112

RESUMO

5,10-methylenetetrahydrofolate reductase (MTHFR) commits folate-derived one-carbon units to generate the methyl-donor S-adenosyl-L-methionine (SAM). Eukaryotic MTHFR appends to the well-conserved catalytic domain (CD) a unique regulatory domain (RD) that confers feedback inhibition by SAM. Here we determine the cryo-electron microscopy structures of human MTHFR bound to SAM and its demethylated product S-adenosyl-L-homocysteine (SAH). In the active state, with the RD bound to a single SAH, the CD is flexible and exposes its active site for catalysis. However, in the inhibited state the RD pocket is remodelled, exposing a second SAM-binding site that was previously occluded. Dual-SAM bound MTHFR demonstrates a substantially rearranged inter-domain linker that reorients the CD, inserts a loop into the active site, positions Tyr404 to bind the cofactor FAD, and blocks substrate access. Our data therefore explain the long-distance regulatory mechanism of MTHFR inhibition, underpinned by the transition between dual-SAM and single-SAH binding in response to cellular methylation status.


Assuntos
Metilenotetra-Hidrofolato Redutase (NADPH2) , S-Adenosilmetionina , Humanos , Regulação Alostérica , Metilenotetra-Hidrofolato Redutase (NADPH2)/química , Microscopia Crioeletrônica , S-Adenosilmetionina/metabolismo , Metilação
16.
Sci Rep ; 14(1): 6915, 2024 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-38519576

RESUMO

Neuronal aging may be, in part, related to a change in DNA methylation. Thus, methyl donors, like folate and methionine, may play a role in cognitive changes associated to neuronal aging. To test the role of these metabolites, we performed stereotaxic microinjection of these molecules into the dentate gyrus (DG) of aged mice (an average age of 21 month). Folate, but not S-Adenosyl-Methionine (SAM), enhances cognition in aged mice. In the presence of folate, we observed partial rejuvenation of DG cells, characterized by the expression of juvenile genes or reorganization of extracellular matrix. Here, we have also tried to identify the mechanism independent of DNA methylation, that involve folate effects on cognition. Our analyses indicated that folate binds to folate receptor α (FRα) and, upon folate binding, FRα is transported to cell nucleus, where it is acting as transcription factor for expressing genes like SOX2 or GluN2B. In this work, we report that a FRα binding peptide also replicates the folate effect on cognition, in aged mice. Our data suggest that such effect is not sex-dependent. Thus, we propose the use of this peptide to improve cognition since it lacks of folate-mediated side effects. The use of synthetic FRα binding peptides emerge as a future strategy for the study of brain rejuvenation.


Assuntos
Receptor 1 de Folato , Rejuvenescimento , Animais , Camundongos , Cognição , Giro Denteado/metabolismo , Receptor 1 de Folato/metabolismo , Ácido Fólico/metabolismo , Metionina , Peptídeos/metabolismo , S-Adenosilmetionina
17.
Microbiol Spectr ; 12(4): e0308623, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38441472

RESUMO

All organisms utilize S-adenosyl-l-methionine (SAM) as a key co-substrate for the methylation of biological molecules, the synthesis of polyamines, and radical SAM reactions. When these processes occur, 5'-deoxy-nucleosides are formed as byproducts such as S-adenosyl-l-homocysteine, 5'-methylthioadenosine (MTA), and 5'-deoxyadenosine (5dAdo). A prevalent pathway found in bacteria for the metabolism of MTA and 5dAdo is the dihydroxyacetone phosphate (DHAP) shunt, which converts these compounds into dihydroxyacetone phosphate and 2-methylthioacetaldehyde or acetaldehyde, respectively. Previous work in other organisms has shown that the DHAP shunt can enable methionine synthesis from MTA or serve as an MTA and 5dAdo detoxification pathway. Rather, the DHAP shunt in Escherichia coli ATCC 25922, when introduced into E. coli K-12, enables the use of 5dAdo and MTA as a carbon source for growth. When MTA is the substrate, the sulfur component is not significantly recycled back to methionine but rather accumulates as 2-methylthioethanol, which is slowly oxidized non-enzymatically under aerobic conditions. The DHAP shunt in ATCC 25922 is active under oxic and anoxic conditions. Growth using 5-deoxy-d-ribose was observed during aerobic respiration and anaerobic respiration with Trimethylamine N-oxide (TMAO), but not during fermentation or respiration with nitrate. This suggests the DHAP shunt may only be relevant for extraintestinal pathogenic E. coli lineages with the DHAP shunt that inhabit oxic or TMAO-rich extraintestinal environments. This reveals a heretofore overlooked role of the DHAP shunt in carbon and energy metabolism from ubiquitous SAM utilization byproducts and suggests a similar role may occur in other pathogenic and non-pathogenic bacteria with the DHAP shunt. IMPORTANCE: The acquisition and utilization of organic compounds that serve as growth substrates are essential for Escherichia coli to grow and multiply. Ubiquitous enzymatic reactions involving S-adenosyl-l-methionine as a co-substrate by all organisms result in the formation of the 5'-deoxy-nucleoside byproducts, 5'-methylthioadenosine and 5'-deoxyadenosine. All E. coli possess a conserved nucleosidase that cleaves these 5'-deoxy-nucleosides into 5-deoxy-pentose sugars for adenine salvage. The DHAP shunt pathway is found in some extraintestinal pathogenic E. coli, but its function in E. coli possessing it has remained unknown. This study reveals that the DHAP shunt enables the utilization of 5'-deoxy-nucleosides and 5-deoxy-pentose sugars as growth substrates in E. coli strains with the pathway during aerobic respiration and anaerobic respiration with TMAO, but not fermentative growth. This provides an insight into the diversity of sugar compounds accessible by E. coli with the DHAP shunt and suggests that the DHAP shunt is primarily relevant in oxic or TMAO-rich extraintestinal environments.


Assuntos
Desoxiadenosinas , Escherichia coli , Metilaminas , S-Adenosilmetionina , Tionucleosídeos , S-Adenosilmetionina/metabolismo , Escherichia coli/metabolismo , Fosfato de Di-Hidroxiacetona , Metionina/metabolismo , Bactérias/metabolismo , Pentoses , Carbono , Açúcares
18.
Chembiochem ; 25(10): e202400079, 2024 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-38477872

RESUMO

S-Adenosyl-l-methionine (SAM) is an important cosubstrate in various biochemical processes, including selective methyl transfer reactions. Simple methods for the (re)generation of SAM analogs could expand the chemistry accessible with SAM-dependent transferases and go beyond methylation reactions. Here we present an efficient enzyme engineering strategy to synthesize different SAM analogs from "off-the-shelf" iodoalkanes through enzymatic alkylation of S-adenosyl-l-homocysteine (SAH). This was achieved by mutating multiple hydrophobic and structurally dynamic amino acids simultaneously. Combinatorial mutagenesis was guided by the natural amino acid diversity and generated a highly functional mutant library. This approach increased the speed as well as the scale of enzyme engineering by providing a panel of optimized enzymes with orders of magnitude higher activities for multiple substrates in just one round of enzyme engineering. The optimized enzymes exhibit catalytic efficiencies up to 31 M-1 s-1, convert various iodoalkanes, including substrates bearing cyclopropyl or aromatic moieties, and catalyze S-alkylation of SAH with very high stereoselectivities (>99 % de). We further report a high throughput chromatographic screening system for reliable and rapid SAM analog analysis. We believe that the methods and enzymes described herein will further advance the field of selective biocatalytic alkylation chemistry by enabling SAM analog regeneration with "off-the-shelf" reagents.


Assuntos
Engenharia de Proteínas , S-Adenosilmetionina , S-Adenosilmetionina/metabolismo , S-Adenosilmetionina/química , Alquilação , Hidrocarbonetos Iodados/química , Biocatálise , Estrutura Molecular
19.
Stem Cells ; 42(5): 475-490, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38427800

RESUMO

Cellular senescence significantly affects the proliferative and differentiation capacities of mesenchymal stem cells (MSCs). Identifying key regulators of senescence and exploring potential intervention strategies, including drug-based approaches, are active areas of research. In this context, S-adenosyl-l-methionine (SAM), a critical intermediate in sulfur amino acid metabolism, emerges as a promising candidate for mitigating MSC senescence. In a hydrogen peroxide-induced MSC aging model (100 µM for 2 hours), SAM (50 and 100 µM) was revealed to alleviate the senescence of MSCs, and also attenuated the level of reactive oxygen species and enhanced the adipogenic and osteogenic differentiation in senescent MSCs. In a premature aging mouse model (subcutaneously injected with 150 mg/kg/day d-galactose in the neck and back for 7 weeks), SAM (30 mg/kg/day by gavage for 5 weeks) was shown to delay the overall aging process while increasing the number and thickness of bone trabeculae in the distal femur. Mechanistically, activation of PI3K/AKT signaling and increased phosphorylation of forkhead box O3 (FOXO3a) was proved to be associated with the antisenescence role of SAM. These findings highlight that the PI3K/AKT/FOXO3a axis in MSCs could play a crucial role in MSCs senescence and suggest that SAM may be a potential therapeutic drug for MSCs senescence and related diseases.


Assuntos
Senescência Celular , Proteína Forkhead Box O3 , Células-Tronco Mesenquimais , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , S-Adenosilmetionina , Transdução de Sinais , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Animais , Senescência Celular/efeitos dos fármacos , Proteína Forkhead Box O3/metabolismo , Proteína Forkhead Box O3/genética , Transdução de Sinais/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , S-Adenosilmetionina/farmacologia , S-Adenosilmetionina/metabolismo , Camundongos , Diferenciação Celular/efeitos dos fármacos , Masculino , Humanos , Camundongos Endogâmicos C57BL
20.
J Am Chem Soc ; 146(10): 6544-6556, 2024 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-38426740

RESUMO

Pyrrolysine, the 22nd amino acid encoded by the natural genetic code, is essential for methanogenic archaea to catabolize methylamines into methane. The structure of pyrrolysine consists of a methylated pyrroline carboxylate that is linked to the ε-amino group of the l-lysine via an amide bond. The biosynthesis of pyrrolysine requires three enzymes: PylB, PylC, and PylD. PylB is a radical S-adenosyl-l-methionine (SAM) enzyme and catalyzes the first biosynthetic step, the isomerization of l-lysine into methylornithine. PylC catalyzes an ATP-dependent ligation of methylornithine and a second l-lysine to form l-lysine-Nε-methylornithine. The last biosynthetic step is catalyzed by PylD via oxidation of the PylC product to form pyrrolysine. While enzymatic reactions of PylC and PylD have been well characterized by X-ray crystallography and in vitro studies, mechanistic understanding of PylB is still relatively limited. Here, we report the first in vitro activity of PylB to form methylornithine via the isomerization of l-lysine. We also identify a lysyl C4 radical intermediate that is trapped, with its electronic structure and geometric structure well characterized by EPR and ENDOR spectroscopy. In addition, we demonstrate that SAM functions as a catalytic cofactor in PylB catalysis rather than canonically as a cosubstrate. This work provides detailed mechanistic evidence for elucidating the carbon backbone rearrangement reaction catalyzed by PylB during the biosynthesis of pyrrolysine.


Assuntos
Lisina , Lisina/análogos & derivados , S-Adenosilmetionina , Lisina/química , Código Genético , Amidas/metabolismo
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