RESUMEN
Successful adaptation of Escherichia coli to constant environmental challenges demands the operation of a wide range of regulatory control mechanisms, some of which are global, while others are specific. Here, we show that the ability of acetate-negative phenotype strains of E. coli devoid of acetate kinase (AK) and phosphotransacetylase (PTA) to assimilate acetate when challenged at the end of growth on acetogenic substrates is explicable by the co-expression of acetyl CoA-synthetase (AcCoA-S) and acetate permease (AP). Furthermore, mRNA transcript measurements for acs and aceA, together with the enzymatic activities of their corresponding enzymes, acetyl CoA synthetase (AcCoA-S) and isocitrate lyase (ICL), clearly demonstrate that the expression of the two enzymes is inextricably linked and triggered in response to growth rate threshold signal (0.4 h-1± 0.03: n4). Interestingly, further restriction of carbon supply to the level of starvation led to the repression of acs (AcCoA-S), ackA (AK) and pta (PTA). Further, we provide evidence that the reaction sequence catalysed by PTA, AK and AcCoA-S is not in operation at low growth rates and that the reaction catalysed by AcCoA-S is not merely an ATP-dissipating reaction but rather advantageous, as it elevates the available free energy (ΔG°) in central metabolism. Moreover, the transcriptomic data reinforce the view that the expression of PEP carboxykinase is essential in gluconeogenic phenotypes.
Asunto(s)
Acetato CoA Ligasa , Escherichia coli , Acetato Quinasa/genética , Acetato Quinasa/metabolismo , Acetato CoA Ligasa/genética , Acetato CoA Ligasa/metabolismo , Acetatos/metabolismo , Acetilcoenzima A/metabolismo , Escherichia coli/metabolismo , Operón , Fosfato Acetiltransferasa/genética , Fosfato Acetiltransferasa/metabolismoRESUMEN
BACKGROUND: Catabolite control protein A (CcpA) regulates the transcription of lactate dehydrogenase and pyruvate formate-lyase in Streptococcus bovis, but knowledge of its role in response to different pH is still limited. In this study, a ccpA-knockout strain of S. bovis S1 was constructed and then used to examine the effects of ccpA gene deletion on the growth and fermentation characteristics of S. bovis S1 at pH 5.5 or 6.5. RESULTS: There was a significant interaction between strain and pH for the maximum specific growth rate (µmax) and growth lag period (λ), which caused a lowest µmax and a longest λ in ccpA-knockout strain at pH 5.5. Deletion of ccpA decreased the concentration and molar percentage of lactic acid, while increased those of formic acid. Strains at pH 5.5 had decreased concentrations of lactic acid and formic acid compared to pH 6.5. The significant interaction between strain and pH caused the highest production of total organic acids and acetic acid in ccpA-knockout strain at pH 6.5. The activities of α-amylase and lactate dehydrogenase decreased in ccpA-knockout strain compared to the wild-type strain, and increased at pH 5.5 compared to pH 6.5. There was a significant interaction between strain and pH for the activity of acetate kinase, which was the highest in the ccpA-knockout strain at pH 6.5. The expression of pyruvate formate-lyase and acetate kinase was higher in the ccpA-knockout strain compared to wild-type strain. The lower pH improved the relative expression of pyruvate formate-lyase, while had no effect on the relative expression of acetate kinase. The strain × pH interaction was significant for the relative expression of lactate dehydrogenase and α-amylase, both of which were highest in the wild-type strain at pH 5.5 and lowest in the ccpA-knockout strain at pH 6.5. CONCLUSIONS: Overall, low pH inhibited the growth of S. bovis S1, but did not affect the fermentation pattern. CcpA regulated S. bovis S1 growth and organic acid fermentation pattern. Moreover, there seemed to be an interaction effect between pH and ccpA deletion on regulating the growth and organic acids production of S. bovis S1.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Represoras/metabolismo , Streptococcus bovis/crecimiento & desarrollo , Streptococcus bovis/metabolismo , Acetato Quinasa/genética , Acetato Quinasa/metabolismo , Acetiltransferasas/metabolismo , Amilasas/genética , Amilasas/metabolismo , Animales , Proteínas Bacterianas/genética , Ácidos Carboxílicos/metabolismo , Fermentación , Regulación Bacteriana de la Expresión Génica , Concentración de Iones de Hidrógeno , L-Lactato Deshidrogenasa/genética , L-Lactato Deshidrogenasa/metabolismo , Mutación , Proteínas Represoras/genética , Rumiantes/microbiologíaRESUMEN
Widely distributed among prokaryotes, short chain fatty acid kinases provide a path for fatty acid entry into central metabolic pathways. These enzymes catalyze the reversible, ATP-dependent synthesis of acyl-phosphates, which leads to the production of acyl-CoA derivatives by a coordinate acyltransferase. To date, characterized representatives of short chain fatty acid kinases exhibit relatively narrow substrate specificity. In this work, biochemical characterization of a predicted acetate kinase from Rhodobacter sphaeroides reveals a novel enzyme with broad substrate specificity for primary fatty acids of varying lengths (C2--C8).
Asunto(s)
Acetato Quinasa/metabolismo , Rhodobacter sphaeroides/enzimología , Acilcoenzima A/metabolismo , Aciltransferasas/metabolismo , Ácidos Grasos/metabolismo , Especificidad por SustratoRESUMEN
Lactobacillus (L.) helveticus is widely used in food industry due to its high proteolytic activity. However, such activity varies greatly between isolates, and the determining factors regulating the strength of proteolytic activity in L. helveticus are unclear. This study sequenced the genomes of 60 fermented food-originated L. helveticus and systemically examined the proteolytic activity-determining factors. Our analyses found that the strength of proteolytic activity in L. helveticus was independent of the isolation source, geographic location, phylogenetic closeness between isolates, and distribution of cell envelope proteinases (CEPs). Genome-wide association study (GWAS) identified two genes, the acetate kinase (ackA) and a hypothetical protein, and 15 single nucleotide polymorphisms (SNPs) that were associated with the strength of the proteolytic activity. Further investigating the functions of these gene components revealed that ackA and two cysteine peptidases coding genes (pepC and srtA) rather than the highly heterogeneous and intraspecific CEPs were linked to the level of proteolytic activity. Moreover, the sequence type (ST) defined by SNP analysis revealed a total of ten STs, and significantly weaker proteolytic activity was observed among isolates of ST2. This study provides practical information for future selection of L. helveticus of strong proteolytic activity.
Asunto(s)
Acetato Quinasa/metabolismo , Proteínas Bacterianas/metabolismo , Productos Lácteos/microbiología , Grano Comestible/microbiología , Alimentos Fermentados/microbiología , Lactobacillus helveticus/enzimología , Péptido Hidrolasas/metabolismo , Acetato Quinasa/química , Acetato Quinasa/genética , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Bovinos , Genoma Bacteriano , Estudio de Asociación del Genoma Completo , Lactobacillus helveticus/genética , Lactobacillus helveticus/aislamiento & purificación , Lactobacillus helveticus/metabolismo , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Filogenia , ProteolisisRESUMEN
The two-component regulatory system CiaRH of Streptococcus pneumoniae affects a large variety of physiological processes including ß-lactam resistance, competence development, maintenance of cell integrity, bacteriocin production, but also host colonization and virulence. The response regulator CiaR is active under a wide variety of conditions and the cognate CiaH kinase is not always needed to maintain CiaR activity. Using tetracycline-controlled expression of ciaR and variants, acetyl phosphate was identified in vivo as the alternative source of CiaR phosphorylation in the absence of CiaH. Concomitant inactivation of ciaH and the acetate kinase gene ackA led to very high levels of CiaR-mediated promoter activation. Strong transcriptional activation was accompanied by a high phosphorylation status of CiaR as determined by Phos-tag gel electrophoresis of S. pneumoniae cell extracts. Furthermore, AckA acted negatively upon acetyl phosphate-dependent phosphorylation of CiaR. Experiments using the Escherichia coli two-hybrid system based on adenylate cyclase reconstitution indicated binding of AckA to CiaR and therefore direct regulation. Subsequent in vitro CiaR phosphorylation experiments confirmed in vivo observations. Purified AckA was able to inhibit acetyl phosphate-dependent phosphorylation. Inhibition required the presence of ADP. AckA-mediated regulation of CiaR phosphorylation is the first example for a regulatory connection of acetate kinase to a response regulator besides controlling acetyl phosphate levels. It will be interesting to see if this novel regulation applies to other response regulators in S. pneumoniae or even in other organisms.
Asunto(s)
Acetato Quinasa/metabolismo , Proteínas Bacterianas/metabolismo , Organofosfatos/metabolismo , Proteínas Quinasas/metabolismo , Streptococcus pneumoniae/metabolismo , Acetato Quinasa/genética , Adenosina Difosfato/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Mutación , Fosforilación , Unión Proteica , Proteínas Quinasas/genética , Transducción de Señal , Streptococcus pneumoniae/genéticaRESUMEN
Acetate is the main by-product from microbial succinate production. In this study, we performed acetate removal by Methanosarcina barkeri 227 for succinate fermentation by Actinobacillus succinogenes 130Z. The acetoclastic methanogen M. barkeri requires similar environmental factors to A. succinogenes, and the conditions required for co-cultivation were optimized in this study: gas used for anaerobicization, strain adaptation, medium composition, pH adjustment, and inoculation time points. M. barkeri 227 was adapted to acetate for 150 days, which accelerated the acetate consumption to 9-fold (from 190 to 1726 mmol gDW-1 day-1). In the acetate-adapted strain, there was a noticeable increase in transcription of genes required for acetoclastic pathway-satP (acetate transporter), ackA (acetate kinase), cdhA (carbon monoxide dehydrogenase/acetyl-CoA synthase complex), and mtrH (methyl-H4STP:CoM methyltransferase), which was not induced before the adaptation process. The activities of two energy-consuming steps in the pathway-acetate uptake and acetate kinase-increased about 3-fold. This acetate-adapted M. barkeri could be successfully applied to succinate fermentation culture of A. succinogenes, but only after pH adjustment following completion of fermentation. This study suggests the utility of M. barkeri as an acetate scavenger during fermentation for further steps towards genetic and process engineering.
Asunto(s)
Acetatos/metabolismo , Actinobacillus/metabolismo , Fermentación , Methanosarcina barkeri/enzimología , Ácido Succínico/metabolismo , Acetato Quinasa/metabolismo , Medios de Cultivo , FosforilaciónRESUMEN
As a multifunctional lactic acid bacterium, Lactobacillus plantarum has been proved to survive in the human gastrointestinal tract, and it can also colonize this tract. In this study, the effects of L. plantarum ATCC 14917 metabolic profile caused by initial acid-base (pH 5.5 and 8.5) stress were investigated using 1 H nuclear magnetic resonance spectroscopy and multivariate data analysis. The results showed that the metabolome mainly consisted of 14 metabolites, including the components like amino acids, sugars, organic acids, and alkaloids. According to the nontargeted principal component analysis, there was a decrease in most of the metabolites in the alkali-treated group (mainly change in PC1) except acetate, whereas the production of lactate and glycine was increased in the acid-treated group (mainly change in PC2). Furthermore, the initial alkali stress inhibits the secretion of lactic acid, as a decrease was observed in the activity of lactate dehydrogenase and acetic dehydrogenase of L. plantarum ATCC 14917 in the alkali group. All these findings revealed that alkali stress could limit the acid environment formation of L. plantarum 14917 in the fermentation process; however, low acid pH is more suitable for the growth of L. plantarum.
Asunto(s)
Ácidos/metabolismo , Álcalis/metabolismo , Lactobacillus plantarum/metabolismo , Estrés Fisiológico , Acetato Quinasa/metabolismo , Proteínas Bacterianas/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Lactobacillus plantarum/enzimología , Lactobacillus plantarum/crecimiento & desarrollo , MetabolomaRESUMEN
BACKGROUND: Pseudomonas putida is a metabolically versatile, genetically accessible, and stress-robust species with outstanding potential to be used as a workhorse for industrial applications. While industry recognises the importance of robustness under micro-oxic conditions for a stable production process, the obligate aerobic nature of P. putida, attributed to its inability to produce sufficient ATP and maintain its redox balance without molecular oxygen, severely limits its use for biotechnology applications. RESULTS: Here, a combination of genome-scale metabolic modelling and comparative genomics is used to pinpoint essential [Formula: see text]-dependent processes. These explain the inability of the strain to grow under anoxic conditions: a deficient ATP generation and an inability to synthesize essential metabolites. Based on this, several P. putida recombinant strains were constructed harbouring acetate kinase from Escherichia coli for ATP production, and a class I dihydroorotate dehydrogenase and a class III anaerobic ribonucleotide triphosphate reductase from Lactobacillus lactis for the synthesis of essential metabolites. Initial computational designs were fine-tuned by means of adaptive laboratory evolution. CONCLUSIONS: We demonstrated the value of combining in silico approaches, experimental validation and adaptive laboratory evolution for microbial design by making the strictly aerobic Pseudomonas putida able to grow under micro-oxic conditions.
Asunto(s)
Proteínas Bacterianas/genética , Microorganismos Modificados Genéticamente , Oxígeno/metabolismo , Pseudomonas putida , Acetato Quinasa/genética , Acetato Quinasa/metabolismo , Anaerobiosis , Proteínas Bacterianas/metabolismo , Dihidroorotato Deshidrogenasa , Escherichia coli/enzimología , Escherichia coli/metabolismo , Genómica , Lactobacillus/enzimología , Lactobacillus/metabolismo , Ingeniería Metabólica , Microorganismos Modificados Genéticamente/genética , Microorganismos Modificados Genéticamente/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Ribonucleótido Reductasas/genética , Ribonucleótido Reductasas/metabolismoRESUMEN
Salmonella enterica catabolizes ethanolamine inside a compartment known as the metabolosome. The ethanolamine utilization (eut) operon of this bacterium encodes all functions needed for the assembly and function of this structure. To date, the roles of EutQ and EutP were not known. Herein we show that both proteins have acetate kinase activity and that EutQ is required during anoxic growth of S. enterica on ethanolamine and tetrathionate. EutP and EutQ-dependent ATP synthesis occurred when enzymes were incubated with ADP, Mg(II) ions and acetyl-phosphate. EutQ and EutP also synthesized acetyl-phosphate from ATP and acetate. Although EutP had acetate kinase activity, ΔeutP strains lacked discernible phenotypes under the conditions where ΔeutQ strains displayed clear phenotypes. The kinetic parameters indicate that EutP is a faster enzyme than EutQ. Our evidence supports the conclusion that EutQ and EutP represent novel classes of acetate kinases. We propose that EutQ is necessary to drive flux through the pathway under physiological conditions, preventing a buildup of acetaldehyde. We also suggest that ATP generated by these enzymes may be used as a substrate for EutT, the ATP-dependent corrinoid adenosyltransferase and for the EutA ethanolamine ammonia-lyase reactivase.
Asunto(s)
Acetato Quinasa/metabolismo , Proteínas Bacterianas/metabolismo , Etanolamina/metabolismo , Salmonella typhimurium/enzimología , Acetato Quinasa/química , Acetato Quinasa/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cinética , Salmonella typhimurium/genética , Salmonella typhimurium/crecimiento & desarrollo , Salmonella typhimurium/metabolismoRESUMEN
Chlamydomonas reinhardtii insertion mutants disrupted for genes encoding acetate kinases (EC 2.7.2.1) (ACK1 and ACK2) and a phosphate acetyltransferase (EC 2.3.1.8) (PAT2, but not PAT1) were isolated to characterize fermentative acetate production. ACK1 and PAT2 were localized to chloroplasts, while ACK2 and PAT1 were shown to be in mitochondria. Characterization of the mutants showed that PAT2 and ACK1 activity in chloroplasts plays a dominant role (relative to ACK2 and PAT1 in mitochondria) in producing acetate under dark, anoxic conditions and, surprisingly, also suggested that Chlamydomonas has other pathways that generate acetate in the absence of ACK activity. We identified a number of proteins associated with alternative pathways for acetate production that are encoded on the Chlamydomonas genome. Furthermore, we observed that only modest alterations in the accumulation of fermentative products occurred in the ack1, ack2, and ack1 ack2 mutants, which contrasts with the substantial metabolite alterations described in strains devoid of other key fermentation enzymes.
Asunto(s)
Acetato Quinasa/metabolismo , Acetatos/metabolismo , Chlamydomonas reinhardtii/enzimología , Cloroplastos/metabolismo , Fosfato Acetiltransferasa/metabolismo , Acetato Quinasa/genética , Proteínas Algáceas/genética , Proteínas Algáceas/metabolismo , Chlamydomonas reinhardtii/genética , Fermentación , Mitocondrias/metabolismo , Mutagénesis Insercional , Fosfato Acetiltransferasa/genéticaRESUMEN
Clostridium tyrobutyricum is a promising organism for butyrate and n-butanol production, but cannot grow on sucrose. Three genes (scrA, scrB, and scrK) involved in the sucrose catabolic pathway, along with an aldehyde/alcohol dehydrogenase gene, were cloned from Clostridium acetobutylicum and introduced into C. tyrobutyricum (Δack) with acetate kinase knockout. In batch fermentation, the engineered strain Ct(Δack)-pscrBAK produced 14.8-18.8 g/L butanol, with a high butanol/total solvent ratio of â¼0.94 (w/w), from sucrose and sugarcane juice. Moreover, stable high butanol production with a high butanol yield of 0.25 g/g and productivity of 0.28 g/Lâh was obtained in batch fermentation without using antibiotics for selection pressure, suggesting that Ct(Δack)-pscrBAK is genetically stable. Furthermore, sucrose utilization by Ct(Δack)-pscrBAK was not inhibited by glucose, which would usually cause carbon catabolite repression on solventogenic clostridia. Ct(Δack)-pscrBAK is thus advantageous for use in biobutanol production from sugarcane juice and other sucrose-rich feedstocks.
Asunto(s)
1-Butanol/metabolismo , Clostridium tyrobutyricum/genética , Clostridium tyrobutyricum/metabolismo , Ingeniería Metabólica , Saccharum/metabolismo , Acetato Quinasa/genética , Acetato Quinasa/metabolismo , Alcohol Deshidrogenasa/genética , Alcohol Deshidrogenasa/metabolismo , Butanoles/metabolismo , Ácido Butírico/metabolismo , Represión Catabólica , Clostridium/genética , Etanol/metabolismo , Fermentación , Jugos de Frutas y Vegetales/microbiología , Expresión Génica , Glucosa/metabolismo , Sacarosa/metabolismoRESUMEN
While many studies have explored the growth of Pseudomonas aeruginosa, comparatively few have focused on its survival. Previously, we reported that endogenous phenazines support the anaerobic survival of P. aeruginosa, yet the physiological mechanism underpinning survival was unknown. Here, we demonstrate that phenazine redox cycling enables P. aeruginosa to oxidize glucose and pyruvate into acetate, which promotes survival by coupling acetate and ATP synthesis through the activity of acetate kinase. By measuring intracellular NAD(H) and ATP concentrations, we show that survival is correlated with ATP synthesis, which is tightly coupled to redox homeostasis during pyruvate fermentation but not during arginine fermentation. We also show that ATP hydrolysis is required to generate a proton-motive force using the ATP synthase complex during fermentation. Together, our results suggest that phenazines enable maintenance of the proton-motive force by promoting redox homeostasis and ATP synthesis. This work demonstrates the more general principle that extracellular redox-active molecules, such as phenazines, can broaden the metabolic versatility of microorganisms by facilitating energy generation.
Asunto(s)
Adenosina Trifosfato/biosíntesis , Metabolismo Energético , Viabilidad Microbiana , Fenazinas/metabolismo , Fuerza Protón-Motriz , Pseudomonas aeruginosa/fisiología , Acetato Quinasa/metabolismo , Ácido Acético/metabolismo , Anaerobiosis , Fermentación , Glucosa/metabolismo , NAD/metabolismo , Oxidación-Reducción , Pseudomonas aeruginosa/metabolismo , Ácido Pirúvico/metabolismoRESUMEN
In the dental caries pathogen Streptococcus mutans, phosphotransacetylase (Pta) catalyzes the conversion of acetyl coenzyme A (acetyl-CoA) to acetyl phosphate (AcP), which can be converted to acetate by acetate kinase (Ack), with the concomitant generation of ATP. A ΔackA mutant displayed enhanced accumulation of AcP under aerobic conditions, whereas little or no AcP was observed in the Δpta or Δpta ΔackA mutant. The Δpta and Δpta ΔackA mutants also had diminished ATP pools compared to the size of the ATP pool for the parental or ΔackA strain. Surprisingly, when exposed to oxidative stress, the Δpta ΔackA strain appeared to regain the capacity to produce AcP, with a concurrent increase in the size of the ATP pool compared to that for the parental strain. The ΔackA and Δpta ΔackA mutants exhibited enhanced (p)ppGpp accumulation, whereas the strain lacking Pta produced less (p)ppGpp than the wild-type strain. The ΔackA and Δpta ΔackA mutants displayed global changes in gene expression, as assessed by microarrays. All strains lacking Pta, which had defects in AcP production under aerobic conditions, were impaired in their abilities to form biofilms when glucose was the growth carbohydrate. Collectively, these data demonstrate the complex regulation of the Pta-Ack pathway and critical roles for these enzymes in processes that appear to be essential for the persistence and pathogenesis of S. mutans.
Asunto(s)
Acetato Quinasa/metabolismo , Acetatos/metabolismo , Redes y Vías Metabólicas/genética , Fosfato Acetiltransferasa/metabolismo , Streptococcus mutans/genética , Streptococcus mutans/metabolismo , Acetato Quinasa/genética , Acetilcoenzima A/metabolismo , Adenosina Trifosfato/metabolismo , Aerobiosis , Biopelículas/crecimiento & desarrollo , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Glucosa/metabolismo , Análisis por Micromatrices , Datos de Secuencia Molecular , Organofosfatos , Estrés Oxidativo , Fosfato Acetiltransferasa/genética , Análisis de Secuencia de ADN , Streptococcus mutans/efectos de los fármacos , Streptococcus mutans/fisiologíaRESUMEN
The genome of the Antarctic bacterium Pseudomonas extremaustralis was analyzed searching for genes involved in environmental adaptability focusing on anaerobic metabolism, osmoregulation, cold adaptation, exopolysaccharide production and degradation of complex compounds. Experimental evidences demonstrated the functionality of several of these pathways, including arginine and pyruvate fermentation, alginate production and growth under cold conditions. Phylogenetic analysis along with genomic island prediction allowed the detection of genes with probable foreign origin such as those coding for acetate kinase, osmotic resistance and colanic acid biosynthesis. These findings suggest that in P. extremaustralis the horizontal transfer events and/or gene redundancy could play a key role in the survival under unfavorable conditions. Comparative genome analysis of these traits in other representative Pseudomonas species highlighted several similarities and differences with this extremophile bacterium.
Asunto(s)
Adaptación Biológica/genética , Genoma Bacteriano , Pseudomonas/genética , Acetato Quinasa/metabolismo , Adenosina Trifosfatasas/química , Alginatos/química , Regiones Antárticas , Arginina/química , Frío , Biología Computacional , Ácidos Cumáricos/química , Ambiente , Fermentación , Ósmosis , Fenotipo , Filogenia , Polisacáridos/química , Pseudomonas/fisiología , Piruvatos/química , Análisis de Secuencia de ADN , Trehalosa/químicaRESUMEN
Recombinant acetate kinase (AcK) was obtained from the aerobic haloalkalitolerant methanotroph Methylomicrobium alcaliphilum 20Z by heterologous expression in Escherichia coli and purification by affinity chromatography. The substrate specificity, the kinetics and oligomeric state of the His6-tagged AcK were determined. The M. alcaliphilum AcK (2 × 45 kDa) catalyzed the reversible phosphorylation of acetate into acetyl phosphate and exhibited a dependence on Mg(2+) or Mn(2+) ions and strong specificity to ATP/ADP. The enzyme showed the maximal activity and high stability at 70 °C. AcK was 20-fold more active in the reaction of acetate synthesis compared to acetate phosphorylation and had a higher affinity to acetyl phosphate (K m 0.11 mM) than to acetate (K m 5.6 mM). The k cat /K m ratios indicated that the enzyme had a remarkably high catalytic efficiency for acetate and ATP formation (k cat/K m = 1.7 × 10(6)) compared to acetate phosphorylation (k cat/K m = 2.5 × 10(3)). The ack gene of M. alcaliphilum 20Z was shown to be co-transcribed with the xfp gene encoding putative phosphoketolase. The Blast analysis revealed the ack and xfp genes in most genomes of the sequenced aerobic methanotrophs, as well as methylotrophic bacteria not growing on methane. The distribution and metabolic role of the postulated phosphoketolase shunted glycolytic pathway in aerobic C1-utilizing bacteria is discussed.
Asunto(s)
Acetato Quinasa/metabolismo , Aldehído-Liasas/metabolismo , Redes y Vías Metabólicas/genética , Methylococcaceae/enzimología , Acetato Quinasa/química , Acetato Quinasa/genética , Cromatografía de Afinidad , Clonación Molecular , Coenzimas/análisis , Estabilidad de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Perfilación de la Expresión Génica , Cinética , Methylococcaceae/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia , Especificidad por Sustrato , TemperaturaRESUMEN
Acetate kinase (ACK) converts acetyl phosphate to acetate along with the generation of ATP in the pathway for mixed-acid fermentation in Lactococcus lactis. The reverse reaction yields acetyl phosphate for assimilation purposes. Remarkably, L. lactis has two ACK isozymes, and the corresponding genes are present in an operon. We purified both enzymes (AckA1 and AckA2) from L. lactis MG1363 and determined their oligomeric state, specific activities, and allosteric regulation. Both proteins form homodimeric complexes, as shown by size exclusion chromatography and static light-scattering measurements. The turnover number of AckA1 is about an order of magnitude higher than that of AckA2 for the reaction in either direction. The Km values for acetyl phosphate, ATP, and ADP are similar for both enzymes. However, AckA2 has a higher affinity for acetate than does AckA1, suggesting an important role under acetate-limiting conditions despite the lower activity. Fructose-1,6-bisphosphate, glyceraldehyde-3-phosphate, and phospho-enol-pyruvate inhibit the activities of AckA1 and AckA2 to different extents. The allosteric regulation of AckA1 and AckA2 and the pool sizes of the glycolytic intermediates are consistent with a switch from homolactic to mixed-acid fermentation upon slowing of the growth rate.
Asunto(s)
Acetato Quinasa/química , Acetato Quinasa/metabolismo , Ácidos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Lactococcus lactis/enzimología , Acetato Quinasa/genética , Regulación Alostérica , Proteínas Bacterianas/genética , Inhibidores Enzimáticos/química , Fermentación , Regulación Enzimológica de la Expresión Génica , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Lactococcus lactis/química , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Especificidad por SustratoRESUMEN
The metabolism of hydroxycinnamic acids by strictly heterofermentative lactic acid bacteria (19 strains) was investigated as a potential alternative energy route. Lactobacillus curvatus PE5 was the most tolerant to hydroxycinnamic acids, followed by strains of Weissella spp., Lactobacillus brevis, Lactobacillus fermentum, and Leuconostoc mesenteroides, for which the MIC values were the same. The highest sensitivity was found for Lactobacillus rossiae strains. During growth in MRS broth, lactic acid bacteria reduced caffeic, p-coumaric, and ferulic acids into dihydrocaffeic, phloretic, and dihydroferulic acids, respectively, or decarboxylated hydroxycinnamic acids into the corresponding vinyl derivatives and then reduced the latter compounds to ethyl compounds. Reductase activities mainly emerged, and the activities of selected strains were further investigated in chemically defined basal medium (CDM) under anaerobic conditions. The end products of carbon metabolism were quantified, as were the levels of intracellular ATP and the NAD(+)/NADH ratio. Electron and carbon balances and theoretical ATP/glucose yields were also estimated. When CDM was supplemented with hydroxycinnamic acids, the synthesis of ethanol decreased and the concentration of acetic acid increased. The levels of these metabolites reflected on the alcohol dehydrogenase and acetate kinase activities. Overall, some biochemical traits distinguished the common metabolism of strictly heterofermentative strains: main reductase activity toward hydroxycinnamic acids, a shift from alcohol dehydrogenase to acetate kinase activities, an increase in the NAD(+)/NADH ratio, and the accumulation of supplementary intracellular ATP. Taken together, the above-described metabolic responses suggest that strictly heterofermentative lactic acid bacteria mainly use hydroxycinnamic acids as external acceptors of electrons.
Asunto(s)
Ácidos Cumáricos/metabolismo , Metabolismo Energético , Lactobacillus/metabolismo , Weissella/metabolismo , Acetato Quinasa/metabolismo , Adenosina Trifosfato/metabolismo , Alcohol Deshidrogenasa/metabolismo , Proteínas Bacterianas/metabolismo , Medios de Cultivo/metabolismo , Transporte de Electrón , Fermentación , Ácido Láctico/metabolismo , Lactobacillus/enzimología , NAD/metabolismo , Weissella/enzimologíaRESUMEN
A process of glucose-6-phosphate (G-6-P) production coupled with an adenosine triphosphate (ATP) regeneration system was constructed that utilized acetyl phosphate (ACP) via acetate kinase (ACKase). The genes glk and ack from Escherichia coli K12 were amplified and cloned into pET-28a(+), then transformed into E. coli BL21 (DE3) and the recombinant strains were named pGLK and pACK respectively. Glucokinase (glkase) in pGLK and ACKase in pACK were both overexpressed in soluble form. G-6-P was efficiently produced from glucose and ACP using a very small amount of ATP. The conversion yield was greater than 97 % when the reaction solution containing 10 mM glucose, 20 mM ACP-Na2, 0.5 mM ATP, 5 mM Mg²âº, 50 mM potassium phosphate buffer (pH 7.0), 4.856 U glkase and 3.632 U ACKase were put into 37 °C water bath for 1 h.
Asunto(s)
Adenosina Trifosfato/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glucoquinasa/metabolismo , Glucosa-6-Fosfato/metabolismo , Ingeniería Metabólica , Acetato Quinasa/genética , Acetato Quinasa/metabolismo , Expresión Génica , Glucosa/metabolismo , Organofosfatos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The green alga Chlamydomonas reinhardtii has numerous genes encoding enzymes that function in fermentative pathways. Among these, the bifunctional alcohol/acetaldehyde dehydrogenase (ADH1), highly homologous to the Escherichia coli AdhE enzyme, is proposed to be a key component of fermentative metabolism. To investigate the physiological role of ADH1 in dark anoxic metabolism, a Chlamydomonas adh1 mutant was generated. We detected no ethanol synthesis in this mutant when it was placed under anoxia; the two other ADH homologs encoded on the Chlamydomonas genome do not appear to participate in ethanol production under our experimental conditions. Pyruvate formate lyase, acetate kinase, and hydrogenase protein levels were similar in wild-type cells and the adh1 mutant, while the mutant had significantly more pyruvate:ferredoxin oxidoreductase. Furthermore, a marked change in metabolite levels (in addition to ethanol) synthesized by the mutant under anoxic conditions was observed; formate levels were reduced, acetate levels were elevated, and the production of CO(2) was significantly reduced, but fermentative H(2) production was unchanged relative to wild-type cells. Of particular interest is the finding that the mutant accumulates high levels of extracellular glycerol, which requires NADH as a substrate for its synthesis. Lactate production is also increased slightly in the mutant relative to the control strain. These findings demonstrate a restructuring of fermentative metabolism in the adh1 mutant in a way that sustains the recycling (oxidation) of NADH and the survival of the mutant (similar to wild-type cell survival) during dark anoxic growth.
Asunto(s)
Alcohol Deshidrogenasa/metabolismo , Chlamydomonas reinhardtii/metabolismo , Glicerol/metabolismo , Proteínas de Plantas/metabolismo , Acetato Quinasa/genética , Acetato Quinasa/metabolismo , Acetatos/metabolismo , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Alcohol Deshidrogenasa/genética , Alcohol Deshidrogenasa/fisiología , Anaerobiosis , Western Blotting , Dióxido de Carbono/metabolismo , Chlamydomonas reinhardtii/enzimología , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/fisiología , Etanol/metabolismo , Fermentación , Formiatos/metabolismo , Genes de Plantas , Hidrógeno/metabolismo , Ácido Láctico/metabolismo , Metaboloma , NAD/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/fisiología , Piruvato-Sintasa/metabolismo , Transcripción GenéticaRESUMEN
Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains a major threat to global health, with the emergence of multi-drug and extensively drug-resistant strains posing a serious challenge. Thereby, understanding the molecular basis of MTB virulence and disease pathogenesis is critical for developing effective therapeutic strategies. Targeting proteins involved in central metabolism has been recognized as a promising therapeutic approach to combat MTB. In this regard, the enzyme AckA of the acetate metabolic pathway which produces acetate from acetyl phosphate, is an important drug target for various pathogenic organisms. Therefore, this study aimed to identify potential AckA inhibitors through in silico methods, including molecular modeling, molecular dynamics simulation (MDS), and high-throughput virtual screening (HTVS) followed by ADMETox, MMGBSA, Density Functional Theory (DFT) calculations. HTVS of one million compounds from the ZINC database against AckA resulted in the top five hits (ZINC82048449, ZINC1219737510, ZINC1771921358, ZINC119699567, and ZINC1427100376) with better binding affinity and optimal binding free energy. MDS studies on complexes revealed that key residues, Asn195, Asp266, Phe267, Gly314, and Asn318 played a significant role in stable interactions of the top-ranked compounds to AckA. These outcomes provide insights into the optimal binding of the leads to inhibit the acetate pathway and aid in the rational design of novel therapeutic agents. Thus, the identified leads may act as promising compounds for targeting AckA and may serve as a potential therapeutic modality for treating TB. Our findings offer valuable insights into the inhibition of the acetate pathway, while also serving as a blueprint for rational drug design. The identified leads hold promise as compelling compounds for targeting AckA, thereby offering a potential therapeutic avenue for tackling TB. Thus, our study uncovers a pathway toward promising TB therapeutics by elucidating AckA inhibitors. By leveraging in silico methodologies, potent compounds that hold the potential to thwart AckA's role in MTB's acetate pathway have been unveiled. This breakthrough fosters optimism in the quest for novel and effective TB treatments, addressing a global health challenge with renewed vigor.