RESUMO
Considered a key taxon in soil and marine microbial communities, myxobacteria exist as coordinated swarms that utilize a combination of lytic enzymes and specialized metabolites to facilitate predation of microbes. This capacity to produce specialized metabolites and the associated abundance of biosynthetic pathways contained within their genomes have motivated continued drug discovery efforts from myxobacteria. Of all myxobacterial biosynthetic gene clusters deposited in the antiSMASH database, only one putative acylhomoserine lactone (AHL) synthase, agpI, was observed, in genome data from Archangium gephyra. Without an AHL receptor also apparent in the genome of A. gephyra, we sought to determine if AgpI was an uncommon example of an orphaned AHL synthase. Herein we report the bioinformatic assessment of AgpI and discovery of a second AHL synthase from Vitiosangium sp. During axenic cultivation conditions, no detectible AHL metabolites were observed in A. gephyra extracts. However, heterologous expression of each synthase in Escherichia coli provided detectible quantities of 3 AHL signals including 2 known AHLs, C8-AHL and C9-AHL. These results suggest that A. gephyra AHL production is dormant during axenic cultivation. The functional, orphaned AHL synthase, AgpI, is unique to A. gephyra, and its utility to the predatory myxobacterium remains unknown.
Assuntos
Acil-Butirolactonas/metabolismo , Ligases/isolamento & purificação , Myxococcales/enzimologia , Acil-Butirolactonas/química , Escherichia coli/genética , Ligases/química , Ligases/genética , Myxococcales/genética , Filogenia , Percepção de Quorum , Análise de Sequência de DNARESUMO
Histidine decarboxylase (HDC; EC 4.1.1.22), an enzyme that catalyzes histamine synthesis with high substrate specificity, is a member of the group II pyridoxal 5'-phosphate (PLP) -dependent decarboxylase family. Tyrosine is a conserved residue among group II PLP-dependent decarboxylases. Human HDC has a Y334 located on a catalytically important loop at the active site. In this study, we demonstrated that a HDC Y334F mutant is capable of catalyzing the decarboxylation-dependent oxidative deamination of histidine to yield imidazole acetaldehyde. Replacement of the active-site Tyr with Phe in group II PLP-dependent decarboxylases, including mammalian aromatic amino acid decarboxylase, plant tyrosine/DOPA decarboxylase, and plant tryptophan decarboxylase, is expected to result in the same functional change, given that a Y-to-F substitution at the corresponding residue (number 260) in the HDC of Morganella morganii, another group II PLP-dependent decarboxylase, yielded the same effect. Thus, it was suggested that the loss of the OH moiety from the active-site Tyr residue of decarboxylase uniquely converts the enzyme to an aldehyde synthase.
Assuntos
Aldeídos/metabolismo , Substituição de Aminoácidos , Histidina Descarboxilase/metabolismo , Imidazóis/metabolismo , Ligases/metabolismo , Aldeído-Desidrogenase Mitocondrial/metabolismo , Catálise , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Histidina Descarboxilase/química , Histidina Descarboxilase/isolamento & purificação , Humanos , Cinética , Ligases/química , Ligases/isolamento & purificação , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrofotometria Ultravioleta , Espectrometria de Massas em TandemRESUMO
Camellia gymnogyna Chang (CgC), a wild tea plant, was discovered on Dayao Mountain, China. However, research regarding this tea plant is limited. Our study found that CgC contains theobromine, caffeine, and theacrine, among which theobromine content was the highest (14.37-39.72â¯mg/g). In addition, theobromine synthase (TS) was partially purified from CgC leaves, up to 35.87-fold, with consecutive chromatography, and its molecular weight was found to be approximately 62â¯kDa. The optimum reaction time, pH, and temperature for theobromine synthase from 7-methylxanthine was found to be 6â¯h, 4, and 45⯰C, respectively. TS expression at both mRNA and protein stages was higher in the first than in the fourth leaf (Pâ¯<â¯0.05). Subcellular localization of TS indicated that it was localized in the nucleus. These results indicate that CgC can be of scientific value and could lead to efficient utilization of this rare wild tea germplasm.
Assuntos
Camellia/enzimologia , Ligases/isolamento & purificação , Teobromina/metabolismo , Alcaloides/metabolismo , Camellia/metabolismo , China , Ligases/metabolismo , Folhas de Planta/químicaRESUMO
Peptides, biologically occurring oligomers of amino acids linked by amide bonds, are essential for living organisms. Many peptides isolated as natural products have biological functions such as antimicrobial, antivirus and insecticidal activities. Peptides often possess structural features or modifications not found in proteins, including the presence of nonproteinogenic amino acids, macrocyclic ring formation, heterocyclization, N-methylation and decoration by sugars or acyl groups. Nature employs various strategies to increase the structural diversity of peptides. Enzymes that modify peptides to yield mature natural products are of great interest for discovering new enzyme chemistry and are important for medicinal chemistry applications. We have discovered novel peptide modifying enzymes and have identified: (i) a new class of amide bond forming-enzymes; (ii) a pathway to biosynthesize a carbonylmethylene-containing pseudodipeptide structure; and (iii) two distinct peptide epimerases. In this review, an overview of our findings on peptide modifying enzymes is presented.
Assuntos
Actinobacteria/enzimologia , Proteínas de Bactérias/metabolismo , Ligases/metabolismo , Biossíntese Peptídica , Processamento de Proteína Pós-Traducional , Racemases e Epimerases/metabolismo , Acilação , Aminoácidos Cíclicos , Proteínas de Bactérias/isolamento & purificação , Biocatálise , Produtos Biológicos/química , Química Farmacêutica , Ciclização , Humanos , Ligases/isolamento & purificação , Metilação , Peptídeos/química , Peptídeos/metabolismo , Peptidomiméticos/química , Peptidomiméticos/metabolismo , Racemases e Epimerases/isolamento & purificaçãoRESUMO
Betalains are plant pigments primarily produced by plants of the order Caryophyllales. Because betalain possesses anti-inflammatory and anticancer activities, it may be useful as a pharmaceutical agent and dietary supplement. Recent studies have identified the genes involved in the betalain biosynthesis of betanin. Amaranthin and celosianin II are abundant in the quinoa (Chenopodium quinoa Willd.) hypocotyl, and amaranthin comprises glucuronic acid bound to betanin; therefore, this suggests the existence of a glucuronyltransferase involved in the synthesis of amaranthin in the quinoa hypocotyl. To identify the gene involved in amaranthin biosynthesis, we performed a BLAST analysis and phylogenetic tree analysis based on sequences homologous to flavonoid glycosyltransferase, followed by expression analysis on the quinoa hypocotyl to obtain three candidate proteins. Production of amaranthin in a transient Nicotiana benthamiana expression system was evaluated for these candidates and one was identified as having the ability to produce amaranthin. The gene encoding this protein was quinoa amaranthin synthetase 1 (CqAmaSy1). We also created a transgenic tobacco bright yellow-2 (BY-2) cell line wherein four betalain biosynthesis genes were introduced to facilitate amaranthin production. This transgenic cell line produced 13.67 ± 4.13 µm (mean ± SEM) amaranthin and 26.60 ± 1.53 µm betanin, whereas the production of isoamaranthin and isobetanin could not be detected. Tests confirmed the ability of amaranthin and betanin to slightly suppress cancer cell viability. Furthermore, amaranthin was shown to significantly inhibit HIV-1 protease activity, whereas betanin did not.
Assuntos
Betacianinas/biossíntese , Chenopodium quinoa/enzimologia , Ligases/isolamento & purificação , Nicotiana/metabolismo , Proteínas de Plantas/isolamento & purificação , Betacianinas/metabolismo , Reatores Biológicos , Células Cultivadas , Chenopodium quinoa/metabolismo , Clonagem Molecular , Protease de HIV , Inibidores da Protease de HIV/metabolismo , Inibidores da Protease de HIV/farmacologia , Ligases/metabolismo , Redes e Vias Metabólicas , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Nicotiana/citologia , Nicotiana/enzimologiaRESUMO
The quorum quenching (QQ) activity of endophytic bacteria associated with medicinal plants was explored. Extracts of the Gram-negative Enterobacter sp. CS66 possessed potent N-acylhomoserine lactone (AHL) hydrolytic activity in vitro. Using degenerate primers, we PCR-amplified an open reading frame (denoted aiiE) from CS66 that was 96% identical to the well-characterised AHL-lactonase AiiA from Bacillus thuringiensis, but only 30% was identical to AHL-lactonases from other Gram-negative species. This confirms that close AiiA homologs can be found in both Gram-positive and Gram-negative bacteria. Purified AiiE exhibited potent AHL-lactonase activity against a broad range of AHLs. Furthermore, aiiE was able to reduce the production of secreted plant cell wall-degrading hydrolytic enzymes when expressed in trans in the economically important plant pathogen, Pectobacterium atrosepticum. Our results indicate the presence of a novel AHL-lactonase in Enterobacter sp. CS66 with significant potential as a biocontrol agent.
Assuntos
Acil-Butirolactonas/metabolismo , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Endófitos/enzimologia , Enterobacter/enzimologia , Ligases/isolamento & purificação , Ligases/metabolismo , Magnoliopsida/microbiologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Endófitos/genética , Endófitos/isolamento & purificação , Endófitos/fisiologia , Enterobacter/genética , Enterobacter/isolamento & purificação , Enterobacter/fisiologia , Cinética , Ligases/química , Ligases/genética , Percepção de Quorum , Alinhamento de SequênciaRESUMO
Breaking and forming peptidyl bonds are fundamental biochemical reactions in protein chemistry. Unlike proteases that are abundantly available, fast-acting ligases are rare. OaAEP1 is an enzyme isolated from the cyclotide-producing plant oldenlandia affinis that displayed weak peptide cyclase activity, despite having a similar structural fold with other asparaginyl endopeptidases (AEP). Here we report the first atomic structure of OaAEP1, at a resolution of 2.56 Å, in its preactivation form. Our structure and biochemical analysis of this enzyme reveals its activation mechanism as well as structural features important for its ligation activity. Importantly, through structure-based mutagenesis of OaAEP1, we obtained an ultrafast variant having hundreds of times faster catalytic kinetics, capable of ligating well-folded protein substrates using only a submicromolar concentration of enzyme. In contrast, the protein-protein ligation activity in the original wild-type OaAEP1 enzyme described previously is extremely weak. Thus, the structure-based engineering of OaAEP1 described here provides a unique and novel recombinant tool that can now be used to conduct various protein labeling and modifications that were extremely challenging before.
Assuntos
Biocatálise , Ligases/metabolismo , Engenharia de Proteínas , Ligases/química , Ligases/isolamento & purificação , Modelos Moleculares , Oldenlandia/enzimologia , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
In this study, 234 bacterial strains were isolated from 7 seaweed species in the Gulf of Mannar on the southeast coast of India. The strains having consistent antimicrobial activity were chosen for further studies, and this constituted about 9.8% of the active strains isolated. Phylogenetic analysis using 16S rDNA sequencing with the help of classical biochemical identification indicated the existence of 2 major phyla, Firmicutes and Proteobacteria. Antimicrobial activity analysis combined with the results of amplifying genes encoding for polyketide synthetase and nonribosomal peptide synthetase showed that seaweed-associated bacteria had broad-spectrum antimicrobial activity. These epibionts might be beneficial to seaweeds by limiting or preventing the development of competing or fouling bacteria. Phylogenetic analysis of ketosynthase (KS) regions with respect to the diverse range of KS domains showed that the KS domains from the candidate isolates were of Type I. The bacterial cultures retained their antimicrobial activities after plasmid curing, which further suggested that the antimicrobial activity of these isolates was not encoded by plasmid, and the genes encoding the antimicrobial product might be present within the genome. Seaweed-associated bacteria with potential antimicrobial activity suggested that the seaweed species are an ideal ecological niche harboring specific bacterial diversity representing a largely underexplored source of antimicrobial secondary metabolites.
Assuntos
Anti-Infecciosos/metabolismo , Bactérias/metabolismo , Proteobactérias/metabolismo , Alga Marinha/microbiologia , Bactérias/genética , Bactérias/isolamento & purificação , Índia , Ligases/isolamento & purificação , Ligases/metabolismo , Filogenia , Proteobactérias/genética , Proteobactérias/isolamento & purificação , Alga Marinha/isolamento & purificaçãoRESUMO
Sponges harbor a remarkable diversity of microbial symbionts in which signal molecules can accumulate and enable cell-cell communication, such as quorum sensing (QS). Bacteria capable of QS were isolated from marine sponges; however, an extremely small fraction of the sponge microbiome is amenable to cultivation. We took advantage of community genome assembly and binning to investigate the uncultured majority of sponge symbionts. We identified a complete N-acyl-homoserine lactone (AHL)-QS system (designated TswIR) and seven partial luxI homologues in the microbiome of Theonella swinhoei. The TswIR system was novel and shown to be associated with an alphaproteobacterium of the order Rhodobacterales, here termed Rhodobacterales bacterium TS309. The tswI gene, when expressed in Escherichia coli, produced three AHLs, two of which were also identified in a T. swinhoei sponge extract. The taxonomic affiliation of the 16S rRNA of Rhodobacterales bacterium TS309 to a sponge-coral specific clade, its enrichment in sponge versus seawater and marine sediment samples, and the presence of sponge-specific features, such as ankyrin-like domains and tetratricopeptide repeats, indicate a likely symbiotic nature of this bacterium.
Assuntos
Alphaproteobacteria/enzimologia , Ligases/isolamento & purificação , Microbiota , Simbiose , Theonella/microbiologia , Acil-Butirolactonas/metabolismo , Alphaproteobacteria/classificação , Alphaproteobacteria/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Oceano Índico , Ligases/genética , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
An enzyme of cephalosporin-acid synthetase produced by the E. coli strain VKPM B-10182 has specificity for the synthesis of ß-lactam antibiotics of the cephalosporin acids class (cefazolin, cefalotin, cefezole etc.). A comparison of the previously determined genomic sequence of E. coli VKPM B-10182 with a genome of the parent E. coli strain ATCC 9637 was performed. Multiple mutations indicating the long selection history of the strain were detected, including mutations in the genes of RNase and ß-lactamases that could enhance the level of enzyme synthesis and reduce the degree of degradation of the synthesized cephalosporin acids. The CASA gene--a direct homolog of the penicillin G-acylase gene--was identified by bioinformatics methods. The homology of the gene was confirmed by gene cloning and the expression and determination of its enzymatic activity in the reaction of cefazolin synthesis. The CASA gene was isolated and cloned into the original expression vector, resulting in an effective E. coli BL2l(DE3) pMD0107 strain producing CASA.
Assuntos
Cefalosporinas/metabolismo , Escherichia coli/enzimologia , Ligases/genética , Clonagem Molecular , Escherichia coli/genética , Genoma Bacteriano , Ligases/isolamento & purificação , Ligases/metabolismo , Penicilina Amidase/genética , Penicilina Amidase/metabolismo , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
This study is the first report of the entire nucleotide sequence of an inositol phosphoceramide synthase gene from the stress-tolerant yeast Pichia kudriavzevii (PkAUR1). Sequence analysis revealed an open reading frame that spans 1,443 bp and encodes a 480-amino-acid-residue protein with the highest sequence similarity (41.7%) to Aur1 from Spathaspora passalidarum. A phenotypic assay with transformed S. cerevisiae and P. kudriavzevii indicated that two amino acid residues, Phe166 and Gly249, play crucial roles in the resistance to aureobasidin A, which is consistent with previous reports for other fungal Aur1s. The GenBank Accession No. for PkAUR1 is KP729614.
Assuntos
Ceramidas/metabolismo , Ligases/isolamento & purificação , Ligases/metabolismo , Organofosfatos/metabolismo , Pichia/enzimologia , Ligases/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Pichia/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Adenosylmethionine synthetase plays a key role in the biogenesis of the sulfonium compound S-adenosylmethionine, the principal widely used methyl donor in the biological methylations. We report here, for the first time, the characterization of adenosylmethionine synthetase from the hyperthermophilic archaeon Pyrococcus furiosus (PfMAT). The gene PF1866 encoding PfMAT was cloned and expressed, and the recombinant protein was purified to homogeneity. PfMAT shares 51, 63, and 82% sequence identity with the homologous enzymes from Sulfolobus solfataricus, Methanococcus jannaschii, and Thermococcus kodakarensis, respectively. PfMAT is a homodimer of 90 kDa highly thermophilic with an optimum temperature of 90 °C and is characterized by remarkable thermodynamic stability (Tm, 99 °C), kinetic stability, and resistance to guanidine hydrochloride-induced unfolding. The latter process is reversible as demonstrated by the analysis of the refolding process by activity assays and fluorescence measurements. Limited proteolysis experiments indicated that the proteolytic cleavage site is localized at Lys148 and that the C-terminal peptide is necessary for the integrity of the active site. PfMAT shows kinetic features that make it the most efficient catalyst for S-adenosylmethionine synthesis among the characterized MAT from Bacteria and Archaea. Molecular and structural characterization of PfMAT could be useful to improve MAT enzyme engineering for biotechnological applications.
Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Ligases/química , Ligases/metabolismo , Metionina/metabolismo , Pyrococcus furiosus/enzimologia , Sequência de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Biocatálise , Clonagem Molecular , Estabilidade Enzimática , Cinética , Ligases/genética , Ligases/isolamento & purificação , Dados de Sequência Molecular , Pyrococcus furiosus/química , Pyrococcus furiosus/genética , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Anthocyanin synthase (ANS), which catalyzes the conversion of colorless leucoanthocyanins into colored anthocyanins, is a key enzyme in the anthocyanin biosynthetic pathway. It plays important roles in plant development and defense. An ANS gene designated as MsANS was cloned from Magnolia sprengeri using rapid amplification of complementary DNA (cDNA) ends technology. The full-length MsANS is 1171-bp long and contains a 1080-bp open reading frame encoding a 360 amino acid polypeptide. In a sequence alignment analysis, the deduced MsANS protein showed high identity to ANS proteins from other plants: Prunus salicina var. cordata (74 % identity), Ampelopsis grossedentata (74 % identity), Pyrus communis (73 % identity), and Prunus avium (73 % identity). A structural analysis showed that MsANS belongs to 2-oxoglutarate (2OG)- and ferrous iron-dependent oxygenase family because it contains three binding sites for 2OG. Real-time quantitative polymerase chain reaction analyses showed that the transcript level of MsANS was 26-fold higher in red petals than in white petals. The accumulation of anthocyanins in petals of white, pink, and red M. sprengeri flowers was analyzed by HPLC. The main anthocyanin was cyanidin-3-o-glucoside chloride, and the red petals contained the highest concentration of this pigment.
Assuntos
Flores/enzimologia , Ligases/genética , Magnolia/enzimologia , Antocianinas/biossíntese , Antocianinas/genética , Cromatografia Líquida de Alta Pressão , Ligases/química , Ligases/isolamento & purificação , Alinhamento de SequênciaRESUMO
Drimenol, a sesquiterpene alcohol, and its derivatives display diverse bio-activities in nature. However, a drimenol synthase gene has yet to be identified. We identified a new sesquiterpene synthase cDNA (VoTPS3) in valerian plant (Valeriana officinalis). Purification and NMR analyses of the VoTPS3-produced terpene, and characterization of the VoTPS3 enzyme confirmed that VoTPS3 synthesizes (-)-drimenol. In feeding assays, possible reaction intermediates, farnesol and drimenyl diphosphate, could not be converted to drimenol, suggesting that the intermediate remains tightly bound to VoTPS3 during catalysis. A mechanistic consideration of (-)-drimenol synthesis suggests that drimenol synthase is likely to use a protonation-initiated cyclization, which is rare for sesquiterpene synthases. VoTPS3 can be used to produce (-)-drimenol, from which useful drimane-type terpenes can be synthesized.
Assuntos
Ligases/genética , Ligases/metabolismo , Terpenos/metabolismo , Valeriana/enzimologia , Clonagem Molecular , Ligases/isolamento & purificação , Sesquiterpenos Policíclicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Valeriana/genéticaRESUMO
Artificial enzymes hold the potential to catalyze valuable reactions not observed in nature. One approach to build artificial enzymes introduces mutations into an existing protein scaffold to enable a new catalytic activity. This process commonly results in a simultaneous reduction of protein stability as an undesired side effect. While protein stability can be increased through techniques like directed evolution, care needs to be taken that added stability, conversely, does not sacrifice the desired activity of the enzyme. Ideally, enzymatic activity and protein stability are engineered simultaneously to ensure that stable enzymes with the desired catalytic properties are isolated. Here, we present the use of the in vitro selection technique mRNA display to isolate enzymes with improved stability and activity in a single step. Starting with a library of artificial RNA ligase enzymes that were previously isolated at ambient temperature and were therefore mostly mesophilic, we selected for thermostable active enzyme variants by performing the selection step at 65 °C. The most efficient enzyme, ligase 10 C, was not only active at 65 °C, but was also an order of magnitude more active at room temperature compared to related enzymes previously isolated at ambient temperature. Concurrently, the melting temperature of ligase 10 C increased by 35 degrees compared to these related enzymes. While low stability and solubility of the previously selected enzymes prevented a structural characterization, the improved properties of the heat-stable ligase 10 C finally allowed us to solve the three-dimensional structure by NMR. This artificial enzyme adopted an entirely novel fold that has not been seen in nature, which was published elsewhere. These results highlight the versatility of the in vitro selection technique mRNA display as a powerful method for the isolation of thermostable novel enzymes.
Assuntos
Ligases/isolamento & purificação , Estabilidade Enzimática , Biblioteca Gênica , Temperatura Alta , Ligases/metabolismo , Estrutura Terciária de Proteína , RNARESUMO
The stringent response is a conserved global regulatory mechanism that is related to the synthesis of (p)ppGpp nucleotides. Gram-positive bacteria, such as Staphylococcus aureus, possess three (p)ppGpp synthases: the bifunctional RSH (RelA/SpoT homolog) protein, which consists of a (p)ppGpp synthase and a (p)ppGpp hydrolase domain, and two truncated (p)ppGpp synthases, designated RelP and RelQ. Here, we characterized these two small (p)ppGpp synthases. Biochemical analyses of purified proteins and in vivo studies revealed a stronger synthetic activity for RelP than for RelQ. However, both enzymes prefer GDP over GTP as the pyrophosphate recipient to synthesize ppGpp. Each of the enzymes was shown to be responsible for the essentiality of the (p)ppGpp hydrolase domain of the RSH protein. The staphylococcal RSH-hydrolase is an efficient enzyme that prevents the toxic accumulation of (p)ppGpp. Expression of (p)ppGpp synthases in a hydrolase-negative background leads not only to growth arrest but also to cell death. Transcriptional analyses showed that relP and relQ are strongly induced upon vancomycin and ampicillin treatments. Accordingly, mutants lacking relP and relQ showed a significantly reduced survival rate upon treatments with cell wall-active antibiotics. Thus, RelP and RelQ are active (p)ppGpp synthases in S. aureus that are induced under cell envelope stress to mediate tolerance against these conditions.
Assuntos
Tolerância a Medicamentos , Regulação Bacteriana da Expressão Gênica , Ligases/metabolismo , Staphylococcus aureus/enzimologia , Staphylococcus aureus/fisiologia , Estresse Fisiológico , Ampicilina/metabolismo , Antibacterianos/metabolismo , Parede Celular/efeitos dos fármacos , Coenzimas/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Ligases/isolamento & purificação , Viabilidade Microbiana/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Vancomicina/metabolismoRESUMO
The chemolithoautotrophic bacterium Nitrosospira multiformis is involved in affecting the process of nitrogen cycling. Here we report the existence and characterization of a functional quorum sensing signal synthase in N. multiformis. One gene (nmuI) playing a role in generating a protein with high levels of similarity to N-acyl homoserine lactone (AHL) synthase protein families was identified. Two AHLs (C14-AHL and 3-oxo-C14-AHL) were detected using an AHL biosensor and liquid chromatography-mass spectrometry (LC-MS) when nmuI, producing a LuxI homologue, was introduced into Escherichia coli. However, by extracting N. multiformis culture supernatants with acidified ethyl acetate, no AHL product was obtained that was capable of activating the biosensor or being detected by LC-MS. According to reverse transcription-PCR, the nmuI gene is transcribed in N. multiformis, and a LuxR homolog (NmuR) in this ammonia-oxidizing strain showed great sensitivity to long-chain AHL signals by solubility assay. A degradation experiment demonstrated that the absence of AHL signals might be attributed to the possible AHL-inactivating activities of this strain. To summarize, an AHL synthase gene (nmuI) acting as a long-chain AHL producer has been found in a chemolithotrophic ammonia-oxidizing microorganism, and the results provide an opportunity to complete the knowledge of the regulatory networks in N. multiformis.
Assuntos
Acil-Butirolactonas/metabolismo , Ligases/isolamento & purificação , Nitrosomonadaceae/enzimologia , Sequência de Aminoácidos , Técnicas Biossensoriais , Cromatografia Líquida , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/genética , Expressão Gênica , Ligases/genética , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
4-Hydroxycoumarin (4HC) type anticoagulants (for example, warfarin) are known to have a significant role in the treatment of thromboembolic diseases--a leading cause of patient morbidity and mortality worldwide. 4HC serves as an immediate precursor of these synthetic anticoagulants. Although 4HC was initially identified as a naturally occurring product, its biosynthesis has not been fully elucidated. Here we present the design, validation, in vitro diagnosis and optimization of an artificial biosynthetic mechanism leading to the microbial biosynthesis of 4HC. Remarkably, function-based enzyme bioprospecting leads to the identification of a characteristic FabH-like quinolone synthase from Pseudomonas aeruginosa with high efficiency on the 4HC-forming reaction, which promotes the high-level de novo biosynthesis of 4HC in Escherichia coli (~500 mg l⻹ in shake flasks) and further in situ semisynthesis of warfarin. This work has the potential to be scaled-up for microbial production of 4HC and opens up the possibility of biosynthesizing diverse coumarin molecules with pharmaceutical importance.
Assuntos
4-Hidroxicumarinas/biossíntese , Anticoagulantes/metabolismo , Proteínas de Bactérias/biossíntese , Ligases/biossíntese , Pró-Fármacos/metabolismo , Pseudomonas aeruginosa/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Escherichia coli/enzimologia , Escherichia coli/genética , Humanos , Cinética , Ligases/genética , Ligases/isolamento & purificação , Engenharia Metabólica , Redes e Vias Metabólicas , Pseudomonas aeruginosa/genética , Varfarina/metabolismoRESUMO
The sulfurtransferase 4-thiouridine synthetase (ThiI) is involved in the ATP-dependent modification of U8 in tRNA. ThiI from Thermotoga maritima was cloned, overexpressed and purified. A complex comprising ThiI and a truncated tRNA was prepared and crystallized, and X-ray diffraction data were collected to a resolution of 3.5 Å. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 102.9, b = 112.8, c = 132.8 Å.
Assuntos
Ligases/química , RNA/química , Thermotoga maritima/enzimologia , Cristalização , Cristalografia por Raios X , Ligases/isolamento & purificação , Ligases/metabolismo , Ligação Proteica , RNA/metabolismo , Tiouridina/metabolismoRESUMO
MbtA is an adenylating enzyme from Mycobacterium tuberculosis that catalyzes the first step in the biosynthesis of the mycobactins. A bisubstrate inhibitor of MbtA (Sal-AMS) was previously described that displays potent antitubercular activity under iron-replete as well as iron-deficient growth conditions. This finding is surprising since mycobactin biosynthesis is not required under iron-replete conditions and suggests off-target inhibition of additional biochemical pathways. As a first step toward a complete understanding of the mechanism of action of Sal-AMS, we have designed and validated an activity-based probe (ABP) for studying Sal-AMS inhibition in M. tuberculosis. This probe labels pure MbtA as well as MbtA in mycobacterial lysate, and labeling can be completely inhibited by preincubation with Sal-AMS. Furthermore, this probe provides a prototypical core scaffold for the creation of ABPs to profile any of the other 66 adenylating enzymes in Mtb or the multitude of adenylating enzymes in other pathogenic bacteria.