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1.
Plant Cell ; 32(4): 820-832, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32060179

RESUMO

Acyl carrier proteins (ACPs) are the scaffolds for fatty acid biosynthesis in living systems, rendering them essential to a comprehensive understanding of lipid metabolism. However, accurate quantitative methods to assess individual acyl-ACPs do not exist. We developed a robust method to quantify acyl-ACPs to the picogram level. We successfully identified acyl-ACP elongation intermediates (3-hydroxyacyl-ACPs and 2,3-trans-enoyl-ACPs) and unexpected medium-chain (C10:1, C14:1) and polyunsaturated long-chain (C16:3) acyl-ACPs, indicating both the sensitivity of the method and how current descriptions of lipid metabolism and ACP function are incomplete. Such ACPs are likely important to medium-chain lipid production for fuels and highlight poorly understood lipid remodeling events in the chloroplast. The approach is broadly applicable to type II fatty acid synthase systems found in plants and bacteria as well as mitochondria from mammals and fungi because it capitalizes on a highly conserved Asp-Ser-Leu-Asp amino acid sequence in ACPs to which acyl groups attach. Our method allows for sensitive quantification using liquid chromatography-tandem mass spectrometry with de novo-generated standards and an isotopic dilution strategy and will fill a gap in our understanding, providing insights through quantitative exploration of fatty acid biosynthesis processes for optimal biofuels, renewable feedstocks, and medical studies in health and disease.

2.
Mol Microbiol ; 113(5): 923-937, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31950558

RESUMO

S-adenosyl-l-methionine (SAM) is a necessary cosubstrate for numerous essential enzymatic reactions including protein and nucleotide methylations, secondary metabolite synthesis and radical-mediated processes. Radical SAM enzymes produce 5'-deoxyadenosine, and SAM-dependent enzymes for polyamine, neurotransmitter and quorum sensing compound synthesis produce 5'-methylthioadenosine as by-products. Both are inhibitory and must be addressed by all cells. This work establishes a bifunctional oxygen-independent salvage pathway for 5'-deoxyadenosine and 5'-methylthioadenosine in both Rhodospirillum rubrum and Extraintestinal Pathogenic Escherichia coli. Homologous genes for this pathway are widespread in bacteria, notably pathogenic strains within several families. A phosphorylase (Rhodospirillum rubrum) or separate nucleoside and kinase (Escherichia coli) followed by an isomerase and aldolase sequentially function to salvage these two wasteful and inhibitory compounds into adenine, dihydroxyacetone phosphate and acetaldehyde or (2-methylthio)acetaldehyde during both aerobic and anaerobic growth. Both SAM by-products are metabolized with equal affinity during aerobic and anaerobic growth conditions, suggesting that the dual-purpose salvage pathway plays a central role in numerous environments, notably the human body during infection. Our newly discovered bifunctional oxygen-independent pathway, widespread in bacteria, salvages at least two by-products of SAM-dependent enzymes for carbon and sulfur salvage, contributing to cell growth.

3.
Plant Methods ; 15: 46, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31110556

RESUMO

Background: Methods used to quantify protein from biological samples are often inaccurate with significant variability that requires care to minimize. The errors result from losses during protein preparation and purification and false detection of interfering compounds or elements. Amino acid analysis (AAA) involves a series of chromatographic techniques that can be used to measure protein levels, avoiding some difficulties and providing specific compositional information. However, unstable derivatives, that are toxic and can be costly, incomplete reactions, inadequate chromatographic separations, and the lack of a single hydrolysis method with sufficient recovery of all amino acids hinder precise protein quantitation using AAA. Results: In this study, a hydrophilic interaction chromatography based method was used to separate all proteinogenic amino acids, including isobaric compounds leucine and isoleucine, prior to detection by multiple reaction monitoring with LC-MS/MS. Through inclusion of commercially available isotopically labeled (13C, 15N) amino acids as internal standards we adapted an isotopic dilution strategy for amino acid-based quantification of proteins. Three hydrolysis methods were tested with ubiquitin, bovine serum albumin, (BSA), and a soy protein biological reference material (SRM 3234; NIST) resulting in protein estimates that were 86-103%, 82-94%, and 90-99% accurate for the three protein samples respectively. The methane sulfonic acid hydrolysis approach provided the best recovery of labile amino acids including: cysteine, methionine and tryptophan that are challenging to accurately quantify. Conclusions: Accurate determination of protein quantity and amino acid composition in heterogeneous biological samples is non-trivial. Recent advances in chromatographic phases and LC-MS/MS based methods, along with the availability of isotopic standards can minimize difficulties in analysis and improve protein quantitation. A robust method is described for high-throughput protein quantification and amino acid compositional analysis. Since accurate measurement of protein quality and quantity are a requirement for many biological studies that relate to crop improvement or more generally, our understanding of metabolism in living systems, we envision this method will have broad applicability.

4.
Appl Environ Microbiol ; 85(13)2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-30979842

RESUMO

ϕEf11 is a temperate Siphoviridae bacteriophage that infects strains of Enterococcus faecalis The ϕEf11 genome, encompassing 65 open reading frames (ORFs), is contained within 42,822 bp of DNA. Within this genome, a module of six lysis-related genes was identified. Based upon sequence homology, one of these six genes, ORF28, was predicted to code for an N-acetylmuramoyl-l-alanine amidase endolysin of 46.133 kDa, composed of 421 amino acids. The PCR-amplified ORF28 was cloned and expressed, and the resulting gene product was affinity purified to homogeneity. The purified protein was obtained from a fusion protein that exhibited a molecular mass of 72.5 kDa, consistent with a 46.1-kDa protein combined with a fused 26.5-kDa glutathione S-transferase tag. It produced rapid, profound lysis in E. faecalis populations and was active against 73 of 103 (71%) E. faecalis strains tested. In addition, it caused substantial destruction of E. faecalis biofilms. The lysin was quite stable, retaining its activity for three years in refrigerated storage, was stable over a wide range of pHs, and was unaffected by the presence of a reducing agent; however, it was inhibited by increasing concentrations of Ca2+ Liquid chromatography-mass spectrometry analysis of E. faecalis cell wall digestion products produced by the ORF28 endolysin indicated that the lysin acted as an N-acetylmuramidase, an endo-ß-N-acetylglucosaminidase, and an endopeptidase, rather than an N-acetylmuramoyl-l-alanine amidase. The ϕEf11 ORF28 lysin shared 10% to 37% amino acid identity with the lytic enzymes of all other characterized E. faecalis bacteriophages.IMPORTANCE The emergence of multidrug-resistant pathogenic microorganisms has brought increasing attention to the urgent need for the development of alternative antimicrobial strategies. One such alternative to conventional antibiotics employs lytic enzymes (endolysins) that are produced by bacteriophages in the course of lytic infection. During lytic infection by a bacteriophage, these enzymes hydrolyze the cell wall peptidoglycan, resulting in the lysis of the host cell. However, external endolysin application can result in lysis from without. In this study, we have cloned, expressed, purified, and characterized an endolysin produced by a bacteriophage infecting strains of Enterococcus faecalis The lysin is broadly active against most of the tested E. faecalis strains and exhibits multifunctional enzymatic specificities that differ from all other characterized endolysins produced by E. faecalis bacteriophages.

5.
Plant Direct ; 1(4): e00018, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31245666

RESUMO

Plant responses to the environment are shaped by external stimuli and internal signaling pathways. In both the model plant Arabidopsis thaliana (Arabidopsis) and crop species, circadian clock factors are critical for growth, flowering, and circadian rhythms. Outside of Arabidopsis, however, little is known about the molecular function of clock gene products. Therefore, we sought to compare the function of Brachypodium distachyon (Brachypodium) and Setaria viridis (Setaria) orthologs of EARLY FLOWERING 3, a key clock gene in Arabidopsis. To identify both cycling genes and putative ELF3 functional orthologs in Setaria, a circadian RNA-seq dataset and online query tool (Diel Explorer) were generated to explore expression profiles of Setaria genes under circadian conditions. The function of ELF3 orthologs from Arabidopsis, Brachypodium, and Setaria was tested for complementation of an elf3 mutation in Arabidopsis. We find that both monocot orthologs were capable of rescuing hypocotyl elongation, flowering time, and arrhythmic clock phenotypes. Using affinity purification and mass spectrometry, our data indicate that BdELF3 and SvELF3 could be integrated into similar complexes in vivo as AtELF3. Thus, we find that, despite 180 million years of separation, BdELF3 and SvELF3 can functionally complement loss of ELF3 at the molecular and physiological level.

6.
Plant Cell ; 28(9): 2026-2042, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27600537

RESUMO

The networks that govern carbon metabolism and control intracellular carbon partitioning in photosynthetic cells are poorly understood. Target of Rapamycin (TOR) kinase is a conserved growth regulator that integrates nutrient signals and modulates cell growth in eukaryotes, though the TOR signaling pathway in plants and algae has yet to be completely elucidated. We screened the unicellular green alga Chlamydomonas reinhardtii using insertional mutagenesis to find mutants that conferred hypersensitivity to the TOR inhibitor rapamycin. We characterized one mutant, vip1-1, that is predicted to encode a conserved inositol hexakisphosphate kinase from the VIP family that pyrophosphorylates phytic acid (InsP6) to produce the low abundance signaling molecules InsP7 and InsP8 Unexpectedly, the rapamycin hypersensitive growth arrest of vip1-1 cells was dependent on the presence of external acetate, which normally has a growth-stimulatory effect on Chlamydomonas. vip1-1 mutants also constitutively overaccumulated triacylglycerols (TAGs) in a manner that was synergistic with other TAG inducing stimuli such as starvation. vip1-1 cells had reduced InsP7 and InsP8, both of which are dynamically modulated in wild-type cells by TOR kinase activity and the presence of acetate. Our data uncover an interaction between the TOR kinase and inositol polyphosphate signaling systems that we propose governs carbon metabolism and intracellular pathways that lead to storage lipid accumulation.

7.
Elife ; 5: e13292, 2016 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-26839287

RESUMO

Plants react to seasonal change in day length through altering physiology and development. Factors that function to harmonize growth with photoperiod are poorly understood. Here we characterize a new protein that associates with both circadian clock and photoreceptor components, named PHOTOPERIODIC CONTROL OF HYPOCOTYL1 (PCH1). pch1 seedlings have overly elongated hypocotyls specifically under short days while constitutive expression of PCH1 shortens hypocotyls independent of day length. PCH1 peaks at dusk, binds phytochrome B (phyB) in a red light-dependent manner, and co-localizes with phyB into photobodies. PCH1 is necessary and sufficient to promote the biogenesis of large photobodies to maintain an active phyB pool after light exposure, potentiating red-light signaling and prolonging memory of prior illumination. Manipulating PCH1 alters PHYTOCHROME INTERACTING FACTOR 4 levels and regulates light-responsive gene expression. Thus, PCH1 is a new factor that regulates photoperiod-responsive growth by integrating the clock with light perception pathways through modulating daily phyB-signaling.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Relógios Circadianos , Luz , Metalochaperonas/metabolismo , Fotoperíodo , Desenvolvimento Vegetal , Transdução de Sinais , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Metalochaperonas/genética , Fitocromo B/metabolismo
8.
Mol Cell Proteomics ; 15(1): 201-17, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26545401

RESUMO

Many species possess an endogenous circadian clock to synchronize internal physiology with an oscillating external environment. In plants, the circadian clock coordinates growth, metabolism and development over daily and seasonal time scales. Many proteins in the circadian network form oscillating complexes that temporally regulate myriad processes, including signal transduction, transcription, protein degradation and post-translational modification. In Arabidopsis thaliana, a tripartite complex composed of EARLY FLOWERING 4 (ELF4), EARLY FLOWERING 3 (ELF3), and LUX ARRHYTHMO (LUX), named the evening complex, modulates daily rhythms in gene expression and growth through transcriptional regulation. However, little is known about the physical interactions that connect the circadian system to other pathways. We used affinity purification and mass spectrometry (AP-MS) methods to identify proteins that associate with the evening complex in A. thaliana. New connections within the circadian network as well as to light signaling pathways were identified, including linkages between the evening complex, TIMING OF CAB EXPRESSION1 (TOC1), TIME FOR COFFEE (TIC), all phytochromes and TANDEM ZINC KNUCKLE/PLUS3 (TZP). Coupling genetic mutation with affinity purifications tested the roles of phytochrome B (phyB), EARLY FLOWERING 4, and EARLY FLOWERING 3 as nodes connecting the evening complex to clock and light signaling pathways. These experiments establish a hierarchical association between pathways and indicate direct and indirect interactions. Specifically, the results suggested that EARLY FLOWERING 3 and phytochrome B act as hubs connecting the clock and red light signaling pathways. Finally, we characterized a clade of associated nuclear kinases that regulate circadian rhythms, growth, and flowering in A. thaliana. Coupling mass spectrometry and genetics is a powerful method to rapidly and directly identify novel components and connections within and between complex signaling pathways.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ritmo Circadiano , Espectrometria de Massas em Tandem/métodos , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cromatografia Líquida , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Transdução de Sinal Luminoso/genética , Microscopia Confocal , Mutação , Plantas Geneticamente Modificadas , Ligação Proteica , Proteoma/genética , Proteoma/metabolismo , Proteômica/métodos , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido
9.
J Biol Chem ; 290(52): 30658-68, 2015 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-26511314

RESUMO

All organisms possess fundamental metabolic pathways to ensure that needed carbon and sulfur compounds are provided to the cell in the proper chemical form and oxidation state. For most organisms capable of using CO2 as sole source of carbon, ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) catalyzes primary carbon dioxide assimilation. In addition, sulfur salvage pathways are necessary to ensure that key sulfur-containing compounds are both available and, where necessary, detoxified in the cell. Using knock-out mutations and metabolomics in the bacterium Rhodospirillum rubrum, we show here that Rubisco concurrently catalyzes key and essential reactions for seemingly unrelated but physiologically essential central carbon and sulfur salvage metabolic pathways of the cell. In this study, complementation and mutagenesis studies indicated that representatives of all known extant functional Rubisco forms found in nature are capable of simultaneously catalyzing reactions required for both CO2-dependent growth as well as growth using 5-methylthioadenosine as sole sulfur source under anaerobic photosynthetic conditions. Moreover, specific inactivation of the CO2 fixation reaction did not affect the ability of Rubisco to support anaerobic 5-methylthioadenosine metabolism, suggesting that the active site of Rubisco has evolved to ensure that this enzyme maintains both key functions. Thus, despite the coevolution of both functions, the active site of this protein may be differentially modified to affect only one of its key functions.


Assuntos
Proteínas de Bactérias/metabolismo , Dióxido de Carbono/metabolismo , Desoxiadenosinas/metabolismo , Redes e Vias Metabólicas , Rhodospirillum rubrum/enzimologia , Ribulose-Bifosfato Carboxilase/química , Ribulose-Bifosfato Carboxilase/metabolismo , Tionucleosídeos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Carbono/metabolismo , Rhodospirillum rubrum/química , Rhodospirillum rubrum/genética , Ribulose-Bifosfato Carboxilase/genética , Enxofre/metabolismo
10.
Proc Natl Acad Sci U S A ; 112(39): 12175-80, 2015 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-26324907

RESUMO

Although natural products have been a particularly rich source of human medicines, activity-based screening results in a very high rate of rediscovery of known molecules. Based on the large number of natural product biosynthetic genes in microbial genomes, many have proposed "genome mining" as an alternative approach for discovery efforts; however, this idea has yet to be performed experimentally on a large scale. Here, we demonstrate the feasibility of large-scale, high-throughput genome mining by screening a collection of over 10,000 actinomycetes for the genetic potential to make phosphonic acids, a class of natural products with diverse and useful bioactivities. Genome sequencing identified a diverse collection of phosphonate biosynthetic gene clusters within 278 strains. These clusters were classified into 64 distinct groups, of which 55 are likely to direct the synthesis of unknown compounds. Characterization of strains within five of these groups resulted in the discovery of a new archetypical pathway for phosphonate biosynthesis, the first (to our knowledge) dedicated pathway for H-phosphinates, and 11 previously undescribed phosphonic acid natural products. Among these compounds are argolaphos, a broad-spectrum antibacterial phosphonopeptide composed of aminomethylphosphonate in peptide linkage to a rare amino acid N(5)-hydroxyarginine; valinophos, an N-acetyl l-Val ester of 2,3-dihydroxypropylphosphonate; and phosphonocystoximate, an unusual thiohydroximate-containing molecule representing a new chemotype of sulfur-containing phosphonate natural products. Analysis of the genome sequences from the remaining strains suggests that the majority of the phosphonate biosynthetic repertoire of Actinobacteria has been captured at the gene level. This dereplicated strain collection now provides a reservoir of numerous, as yet undiscovered, phosphonate natural products.


Assuntos
Actinobacteria/química , Actinobacteria/genética , Produtos Biológicos/química , Descoberta de Drogas/tendências , Genoma Bacteriano/genética , Genômica/métodos , Ácidos Fosforosos/análise , Sequência de Bases , Descoberta de Drogas/métodos , Biblioteca Gênica , Genômica/tendências , Dados de Sequência Molecular , Análise de Sequência de DNA
11.
Plant J ; 82(6): 991-1003, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25939370

RESUMO

Steroid alkaloids have been shown to elicit a wide range of pharmacological effects that include anticancer and antifungal activities. Understanding the biosynthesis of these molecules is essential to bioengineering for sustainable production. Herein, we investigate the biosynthetic pathway to cyclopamine, a steroid alkaloid that shows promising antineoplastic activities. Supply of cyclopamine is limited, as the current source is solely derived from wild collection of the plant Veratrum californicum. To elucidate the early stages of the pathway to cyclopamine, we interrogated a V. californicum RNA-seq dataset using the cyclopamine accumulation profile as a predefined model for gene expression with the pattern-matching algorithm Haystack. Refactoring candidate genes in Sf9 insect cells led to discovery of four enzymes that catalyze the first six steps in steroid alkaloid biosynthesis to produce verazine, a predicted precursor to cyclopamine. Three of the enzymes are cytochromes P450 while the fourth is a γ-aminobutyrate transaminase; together they produce verazine from cholesterol.


Assuntos
Enzimas/metabolismo , Alcaloides de Veratrum/metabolismo , Veratrum/genética , Veratrum/metabolismo , 4-Aminobutirato Transaminase/genética , 4-Aminobutirato Transaminase/metabolismo , Algoritmos , Animais , Vias Biossintéticas , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Enzimas/genética , Perfilação da Expressão Gênica/métodos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Análise de Sequência de RNA/métodos , Células Sf9 , Transcriptoma
12.
ACS Synth Biol ; 4(2): 132-40, 2015 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-24377365

RESUMO

Engineering metabolic biosynthetic pathways has enabled the microbial production of many useful chemicals. However, pathway productivities and yields are often limited by metabolic imbalances. Synthetic regulatory circuits have been shown to be able to balance engineered pathways, improving titers and productivities. Here we developed a negative feedback regulatory circuit based on a malonyl-CoA-based sensor-actuator. Malonyl-CoA is biosynthesized from acetyl-CoA by the acetyl-CoA carboxylase, which is the rate-limiting step for fatty acid biosynthesis. Overexpression of acetyl-CoA carboxylase improves fatty acid production, but slows down cell growth. We have devised a malonyl-CoA sensor-actuator that controls gene expression levels based on intracellular malonyl-CoA concentrations. This sensor-actuator is used to construct a negative feedback circuit to regulate the expression of acetyl-CoA carboxylase. The negative feedback circuit is able to up-regulate acetyl-CoA carboxylase expression when the malonyl-CoA concentration is low and down-regulate acetyl-CoA carboxylase expression when excess amounts of malonyl-CoA have accumulated. We show that the regulatory circuit effectively alleviates the toxicity associated with acetyl-CoA carboxylase overexpression. When used to regulate the fatty acid pathway, the feedback circuit increases fatty acid titer and productivity by 34% and 33%, respectively.


Assuntos
Ácidos Graxos/biossíntese , Malonil Coenzima A/metabolismo , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Escherichia coli/metabolismo , Ácidos Graxos/análise , Engenharia Genética , Malonil Coenzima A/análise , Espectrometria de Massas , Engenharia Metabólica , Regiões Promotoras Genéticas
13.
J Bacteriol ; 196(9): 1768-79, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24584498

RESUMO

Two related actinomycetes, Glycomyces sp. strain NRRL B-16210 and Stackebrandtia nassauensis NRRL B-16338, were identified as potential phosphonic acid producers by screening for the gene encoding phosphoenolpyruvate (PEP) mutase, which is required for the biosynthesis of most phosphonates. Using a variety of analytical techniques, both strains were subsequently shown to produce phosphonate-containing exopolysaccharides (EPS), also known as phosphonoglycans. The phosphonoglycans were purified by sequential organic solvent extractions, methanol precipitation, and ultrafiltration. The EPS from the Glycomyces strain has a mass of 40 to 50 kDa and is composed of galactose, xylose, and five distinct partially O-methylated galactose residues. Per-deutero-methylation analysis indicated that galactosyl residues in the polysaccharide backbone are 3,4-linked Gal, 2,4-linked 3-MeGal, 2,3-linked Gal, 3,6-linked 2-MeGal, and 4,6-linked 2,3-diMeGal. The EPS from the Stackebrandtia strain is comprised of glucose, galactose, xylose, and four partially O-methylated galactose residues. Isotopic labeling indicated that the O-methyl groups in the Stackebrandtia phosphonoglycan arise from S-adenosylmethionine. The phosphonate moiety in both phosphonoglycans was shown to be 2-hydroxyethylphosphonate (2-HEP) by (31)P nuclear magnetic resonance (NMR) and mass spectrometry following strong acid hydrolysis of the purified molecules. Partial acid hydrolysis of the purified EPS from Glycomyces yielded 2-HEP in ester linkage to the O-5 or O-6 position of a hexose and a 2-HEP mono(2,3-dihydroxypropyl)ester. Partial acid hydrolysis of Stackebrandtia EPS also revealed the presence of 2-HEP mono(2,3-dihydroxypropyl)ester. Examination of the genome sequences of the two strains revealed similar pepM-containing gene clusters that are likely to be required for phosphonoglycan synthesis.


Assuntos
Actinomycetales/química , Organofosfonatos/metabolismo , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/isolamento & purificação , Actinomycetales/enzimologia , Actinomycetales/genética , Actinomycetales/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Carboidratos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Peso Molecular , Fosfotransferases (Fosfomutases)/genética , Fosfotransferases (Fosfomutases)/metabolismo , Polissacarídeos Bacterianos/metabolismo
14.
PLoS One ; 9(3): e91537, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24626471

RESUMO

Phenotype in multicellular organisms is the consequence of dynamic metabolic events that occur in a spatially dependent fashion. This spatial and temporal complexity presents challenges for investigating metabolism; creating a need for improved methods that effectively probe biochemical events such as amino acid biosynthesis. Isotopic labeling can provide a temporal-spatial recording of metabolic events through, for example, the description of enriched amino acids in the protein pool. Proteins are therefore an important readout of metabolism and can be assessed with modern mass spectrometers. We compared the measurement of isotopic labeling in MS2 spectra obtained from tandem mass spectrometry under either higher energy collision dissociation (HCD) or collision induced dissociation (CID) at varied energy levels. Developing soybean embryos cultured with or without 13C-labeled substrates, and Escherichia coli MG1655 enriched by feeding 7% uniformly labeled glucose served as a source of biological material for protein evaluation. CID with low energies resulted in a disproportionate amount of heavier isotopologues remaining in the precursor isotopic distribution. HCD resulted in fewer quantifiable products; however deviation from predicted distributions were small relative to the CID-based comparisons. Fragment ions have the potential to provide information on the labeling of amino acids in peptides, but our results indicate that without further development the use of this readout in quantitative methods such as metabolic flux analysis is limited.


Assuntos
Escherichia coli/química , Isótopos/química , Soja/química , Espectrometria de Massas em Tandem , Aminoácidos/química , Cromatografia Líquida , Marcação por Isótopo , Espectrometria de Massas , Fragmentos de Peptídeos/química , Peptídeos/química , Fenótipo
15.
J Biol Chem ; 289(9): 5412-6, 2014 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-24448804

RESUMO

Cancer cells are characterized by elevated levels of reactive oxygen species, which are produced mainly by the mitochondria. The dismutase SOD2 localizes in the matrix and is a major antioxidant. The activity of SOD2 is regulated by the deacetylase SIRT3. Recent studies indicated that SIRT3 is decreased in 87% of breast cancers, implying that the activity of SOD2 is compromised. The resulting elevation in reactive oxygen species was shown to be essential for the metabolic reprograming toward glycolysis. Here, we show that SOD2 itself is down-regulated in breast cancer cell lines. Further, activation of oncogenes, such as Ras, promotes the rapid down-regulation of SOD2. Because in the absence of SOD2, superoxide levels are elevated in the matrix, we reasoned that mechanisms must exist to retain low levels of superoxide in other cellular compartments especially in the intermembrane space of the mitochondrial to avoid irreversible damage. The dismutase SOD1 also acts as an antioxidant, but it localizes to the cytoplasm and the intermembrane space of the mitochondria. We report here that loss of SOD2 correlates with the overexpression of SOD1. Further, we show that mitochondrial SOD1 is the main dismutase activity in breast cancer cells but not in non-transformed cells. In addition, we show that the SOD1 inhibitor LCS-1 leads to a drastic fragmentation and swelling of the matrix, suggesting that in the absence of SOD2, SOD1 is required to maintain the integrity of the organelle. We propose that by analogy to the cadherin switch during epithelial-mesenchymal transition, cancer cells also undergo a SOD switch during transformation.


Assuntos
Neoplasias da Mama/enzimologia , Transição Epitelial-Mesenquimal , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/metabolismo , Superóxido Dismutase/biossíntese , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Sirtuína 3/genética , Sirtuína 3/metabolismo , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Superóxidos/metabolismo
16.
J Nat Prod ; 77(2): 243-9, 2014 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-24437999

RESUMO

Streptomyces regensis strain WC-3744 was identified as a potential phosphonic acid producer in a large-scale screen of microorganisms for the presence of the pepM gene, which encodes the key phosphonate biosynthetic enzyme phosphoenolpyruvate phosphonomutase. (31)P NMR revealed the presence of several unidentified phosphonates in spent medium after growth of S. regensis. These compounds were purified and structurally characterized via extensive 1D and 2D NMR spectroscopic and mass spectrometric analyses. Three new phosphonic acid metabolites, whose structures were confirmed by comparison to chemically synthesized standards, were observed: (2-acetamidoethyl)phosphonic acid (1), (2-acetamido-1-hydroxyethyl)phosphonic (3), and a novel cyanohydrin-containing phosphonate, (cyano(hydroxy)methyl)phosphonic acid (4). The gene cluster responsible for synthesis of these molecules was also identified from the draft genome sequence of S. regensis, laying the groundwork for future investigations into the metabolic pathway leading to this unusual natural product.


Assuntos
Produtos Biológicos/isolamento & purificação , Nitrilos/isolamento & purificação , Organofosfonatos/isolamento & purificação , Streptomyces , Sequência de Bases , Produtos Biológicos/química , Dados de Sequência Molecular , Estrutura Molecular , Nitrilos/química , Ressonância Magnética Nuclear Biomolecular , Organofosfonatos/química , Streptomyces/química , Streptomyces/enzimologia , Streptomyces/genética
17.
Metabolomics ; 2014(August)2014 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-25705145

RESUMO

While recent advances in metabolomic measurement technologies have been dramatic, extracting biological insight from complex metabolite profiles remains a challenge. We present an analytical strategy that uses data obtained from high resolution liquid chromatography-mass spectrometry and a bioinformatics toolset for detecting actively changing metabolic pathways upon external perturbation. We begin with untargeted metabolite profiling to nominate altered metabolites and identify pathway candidates, followed by validation of those pathways with transcriptomics. Using the model organisms Rhodospirillum rubrum and Bacillus subtilis, our results reveal metabolic pathways that are interconnected with methionine salvage. The rubrum-type methionine salvage pathway is interconnected with the active methyl cycle in which re-methylation, a key reaction for recycling methionine from homocysteine, is unexpectedly suppressed; instead, homocysteine is catabolized by the transsulfuration pathway. Notably, the non-mevalonate pathway is repressed, whereas the rubrum-type methionine salvage pathway contributes to isoprenoid biosynthesis upon 5'-methylthioadenosine feeding. In this process, glutathione functions as a coenzyme in vivo when 1-methylthio-d-xylulose 5-phosphate (MTXu 5-P) methylsulfurylase catalyzes dethiomethylation of MTXu 5-P. These results clearly show that our analytical approach enables unexpected metabolic pathways to be uncovered.

18.
Angew Chem Int Ed Engl ; 53(5): 1334-7, 2014 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-24376039

RESUMO

Natural product discovery has been boosted by genome mining approaches, but compound purification is often still challenging. We report an enzymatic strategy for "stable isotope labeling of phosphonates in extract" (SILPE) that facilitates their purification. We used the phosphonate methyltransferase DhpI involved in dehydrophos biosynthesis to methylate a variety of phosphonate natural products in crude spent medium with a mixture of labeled and unlabeled S-adenosyl methionine. Mass-guided fractionation then allowed straightforward purification. We illustrate its utility by purifying a phosphonate that led to the identification of the fosfazinomycin biosynthetic gene cluster. This unusual natural product contains a hydrazide linker between a carboxylic acid and a phosphonic acid. Bioinformatic analysis of the gene cluster provides insights into how such a structure might be assembled.


Assuntos
Antibacterianos/biossíntese , Produtos Biológicos/metabolismo , Hidrazinas/síntese química , Metiltransferases/metabolismo , Organofosfonatos/química , Compostos Organofosforados/síntese química , Antibacterianos/química , Produtos Biológicos/química , Biologia Computacional , DNA Fúngico/genética , Hidrazinas/química , Hidrazinas/metabolismo , Marcação por Isótopo , Metiltransferases/genética , Família Multigênica , Fases de Leitura Aberta/genética , Compostos Organofosforados/química , S-Adenosilmetionina/química , Streptomyces/genética , Streptomyces/metabolismo
19.
ACS Chem Biol ; 8(5): 908-13, 2013 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-23474169

RESUMO

Naturally occurring phosphonates such as phosphinothricin (Glufosinate, a commercially used herbicide) and fosfomycin (Monurol, a clinically used antibiotic) have proved to be potent and useful biocides. Yet this class of natural products is still an under explored family of secondary metabolites. Discovery of the biosynthetic pathways responsible for the production of these compounds has been simplified by using gene based screening approaches, but detection and identification of the natural products the genes produce have been hampered by a lack of high-throughput methods for screening potential producers under various culture conditions. Here, we present an efficient mass-spectrometric method for the selective detection of natural products containing phosphonate and phosphinate functional groups. We have used this method to identify a new phosphonate metabolite, phosacetamycin, whose structure, biological activity, and biosynthetic gene cluster are reported.


Assuntos
Acetamidas/análise , Acetamidas/farmacologia , Antibacterianos/química , Antibacterianos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Espectrometria de Massas/métodos , Ácidos Fosforosos/análise , Ácidos Fosforosos/farmacologia , Streptomyces/genética , Acetamidas/química , Antibacterianos/análise , Vias Biossintéticas/genética , Cromatografia de Afinidade/métodos , Escherichia coli/efeitos dos fármacos , Genoma Bacteriano , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Estrutura Molecular , Família Multigênica , Fosfatos/isolamento & purificação , Ácidos Fosforosos/química , Streptomyces/metabolismo
20.
Biochemistry ; 51(42): 8324-6, 2012 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-23035785

RESUMO

Rhodospirillum rubrum produces 5-methylthioadenosine (MTA) from S-adenosylmethionine in polyamine biosynthesis; however, R. rubrum lacks the classical methionine salvage pathway. Instead, MTA is converted to 5-methylthio-d-ribose 1-phosphate (MTR 1-P) and adenine; MTR 1-P is isomerized to 1-methylthio-d-xylulose 5-phosphate (MTXu 5-P) and reductively dethiomethylated to 1-deoxy-d-xylulose 5-phosphate (DXP), an intermediate in the nonmevalonate isoprenoid pathway [Erb, T. J., et al. (2012) Nat. Chem. Biol., in press]. Dethiomethylation, a novel route to DXP, is catalyzed by MTXu 5-P methylsulfurylase. An active site Cys displaces the enolate of DXP from MTXu 5-P, generating a methyl disulfide intermediate.


Assuntos
Pentosefosfatos/biossíntese , Rhodospirillum rubrum/metabolismo , Sulfurtransferases/metabolismo , Redes e Vias Metabólicas , Ressonância Magnética Nuclear Biomolecular , Pentosefosfatos/metabolismo , Ribosemonofosfatos/metabolismo , Tioglicosídeos/metabolismo
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