RESUMEN
Coronatine and related bacterial phytotoxins are mimics of the hormone jasmonyl-L-isoleucine (JA-Ile), which mediates physiologically important plant signalling pathways1-4. Coronatine-like phytotoxins disrupt these essential pathways and have potential in the development of safer, more selective herbicides. Although the biosynthesis of coronatine has been investigated previously, the nature of the enzyme that catalyses the crucial coupling of coronafacic acid to amino acids remains unknown1,2. Here we characterize a family of enzymes, coronafacic acid ligases (CfaLs), and resolve their structures. We found that CfaL can also produce JA-Ile, despite low similarity with the Jar1 enzyme that is responsible for ligation of JA and L-Ile in plants5. This suggests that Jar1 and CfaL evolved independently to catalyse similar reactions-Jar1 producing a compound essential for plant development4,5, and the bacterial ligases producing analogues toxic to plants. We further demonstrate how CfaL enzymes can be used to synthesize a diverse array of amides, obviating the need for protecting groups. Highly selective kinetic resolutions of racemic donor or acceptor substrates were achieved, affording homochiral products. We also used structure-guided mutagenesis to engineer improved CfaL variants. Together, these results show that CfaLs can deliver a wide range of amides for agrochemical, pharmaceutical and other applications.
Asunto(s)
Amidas/metabolismo , Ligasas/química , Ligasas/metabolismo , Amidas/química , Aminoácidos/biosíntesis , Aminoácidos/química , Azospirillum lipoferum/enzimología , Azospirillum lipoferum/genética , Ácidos Carboxílicos/metabolismo , Ciclopentanos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Herbicidas/química , Herbicidas/metabolismo , Indenos/química , Isoleucina/análogos & derivados , Isoleucina/biosíntesis , Isoleucina/química , Cinética , Modelos Moleculares , Pectobacterium/enzimología , Pectobacterium/genética , Pseudomonas syringae/enzimología , Pseudomonas syringae/genéticaAsunto(s)
Azospirillum/enzimología , Monofenol Monooxigenasa/aislamiento & purificación , Monofenol Monooxigenasa/metabolismo , Azospirillum/crecimiento & desarrollo , Azospirillum brasilense/enzimología , Azospirillum brasilense/crecimiento & desarrollo , Azospirillum lipoferum/enzimología , Azospirillum lipoferum/crecimiento & desarrollo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Electroforesis en Gel de Poliacrilamida , Peso Molecular , Monofenol Monooxigenasa/químicaRESUMEN
The present study relates to a nanotechnology enabled method in which purified laccase from Escherichia coli AKL2 was supplemented with 100 µM copper oxide nanoparticles (Cu(2)O) (NP-laccase). The activity, half life and stability of NP-laccase were enhanced by 4, 42 and 36-fold respectively at high temperature (80 °C) and also over a wide range of pH (4-12) than laccase (in the presence of 0.18 mM CuSO(4)). Thermodynamic analysis of the nanoparticle-induced enzyme stability revealed an enhanced entropy-enthalpy compensation at 80 °C, which reflected the maintenance of its native structure. This was further supported by CD studies. The enhanced activity and thermostability of NP-laccase can be utilized for efficient decolorisation of dyes (both phenolic and azo).
Asunto(s)
Azospirillum lipoferum/enzimología , Colorantes/metabolismo , Cobre/metabolismo , Lacasa/metabolismo , Nanopartículas del Metal , Eliminación de Residuos Líquidos/métodos , Contaminantes Químicos del Agua/metabolismo , Purificación del Agua/métodos , Azospirillum lipoferum/genética , Secuencia de Bases , Dicroismo Circular , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , ARN Ribosómico 16S/genética , Ribotipificación , Análisis de Secuencia de ADN , TemperaturaRESUMEN
The phytostimulatory alphaproteobacterium Azospirillum lipoferum 4B exhibits the plant-beneficial gene acdS, which enables deamination of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). Here, we show that acdS is in the vicinity of acdR, a homolog to leucine-responsive regulator lrp, in A. lipoferum 4B and most other acdS+ Proteobacteria. Unlike in Beta- and Gammaproteobacteria, acdS (and acdR) is preferentially located on symbiotic islands and plasmids in Alphaproteobacteria. In A. lipoferum 4B, acdS was mapped on a 750-kb plasmid that is lost during phenotypic variation, whereas other phytobeneficial genes such as nifH (associative nitrogen fixation) are maintained. In Proteobacteria, the phylogenies of acdR and acdS were largely but not totally congruent, despite physical proximity of the genes, regardless of whether DNA or deduced protein sequences were used. Potential Lrp, cAMP receptor protein (CRP) and fumarate-nitrate reduction regulator (FNR) binding sites were evidenced in the acdS promoter regions of strain 4B and most of 46 other acdS+ Proteobacteria. Indeed, transcriptional and enzymatic analyses done in vitro pointed to the involvement of Lrp- and FNR-like transcriptional up-regulation of ACC deaminase activity in A. lipoferum 4B. This is the first synteny, phylogenetic, and functional analysis of factors modulating acdS expression in Azospirillum plant growth-promoting rhizobacterium.
Asunto(s)
Azospirillum lipoferum , Liasas de Carbono-Carbono , Regulación Bacteriana de la Expresión Génica , Proteína Reguladora de Respuesta a la Leucina , Filogenia , Proteobacteria , Secuencia de Aminoácidos , Aminoácidos Cíclicos/metabolismo , Azospirillum lipoferum/enzimología , Azospirillum lipoferum/genética , Azospirillum lipoferum/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Liasas de Carbono-Carbono/genética , Liasas de Carbono-Carbono/metabolismo , Regulación Enzimológica de la Expresión Génica , Leucina/metabolismo , Proteína Reguladora de Respuesta a la Leucina/química , Proteína Reguladora de Respuesta a la Leucina/genética , Proteína Reguladora de Respuesta a la Leucina/metabolismo , Datos de Secuencia Molecular , Fenotipo , Proteobacteria/clasificación , Proteobacteria/enzimología , Proteobacteria/genética , Proteobacteria/metabolismo , Alineación de SecuenciaRESUMEN
AIMS: The objective of this paper was to study the adaptation dynamics and biochemical response of Azospirillum lipoferum grown in a continuous culture at various environmental shifts. METHODS AND RESULTS: The kinetics of A. lipoferum Sp 59b grown at steady states in a microaerobic chemostatic environment deviated from a typical Monod kinetics in both low and high dilution rates (D) due to several metabolic shifts that occurred in the microbial cell. When NH4Cl was exhausted (at low D), the microbial cell partitioned carbon flow in order to sustain growth, nitrogen fixation and assimilation processes (occurred via the glutamate synthase reaction). Increasing D the specific activities of the enzymes involved in the tricarboxylic acid cycle and the respiration rate were decreased. At transitory states, under optimal for nitrogen fixation dissolved oxygen (DO) concentrations, ammonium nitrogen negatively affected, besides nitrogen fixing activity, the bacterial growth. At sub-optimal for nitrogen fixation DO concentration (i.e. 1.56 microM) and 0.1 g l(-1) NH4Cl in the fed medium, the activities of citrate synthase and succinate dehydrogenase were significantly reduced. CONCLUSIONS: Important shifts in both carbon and nitrogen metabolism occur in A. lipoferum grown in the presence of the ammonium nitrogen, while the boundaries of ammonium nitrogen concentration in which A. lipoferum can be adapted depend on the DO concentration in the growth environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Studies on growth dynamics and physiology of A. lipoferum, grown in experimental model systems, can contribute to an efficient application of these bacteria as plant-growth-promoting-agents.
Asunto(s)
Cloruro de Amonio/metabolismo , Azospirillum lipoferum/crecimiento & desarrollo , Aerobiosis/fisiología , Azospirillum lipoferum/enzimología , Carbono/metabolismo , Citrato (si)-Sintasa/metabolismo , Medios de Cultivo , Glutamato Sintasa/metabolismo , Isocitrato Deshidrogenasa/metabolismo , Modelos Biológicos , Nitrógeno/metabolismo , Fijación del Nitrógeno/fisiología , Oxígeno/metabolismo , Polisacáridos Bacterianos/metabolismo , Succinato Deshidrogenasa/metabolismoRESUMEN
The utilization of some agro-industrial wastes as soil conditioners to provide free-living nitrogen-fixing bacterial populations (e.g. Azospirillum spp.) with carbon and energy sources, may be an interesting perspective for agriculture. However, the presence of ammonium nitrogen in cultivated soils and/or various wastes could inhibit the growth of the nitrogen-fixing populations. The present investigation shows that growth of Azospirillum lipoferum was restricted at a dissolved oxygen (DO) concentration equal to 135 microM, when the initial NH4Cl concentration increased from 0.5 to 0.9 g/l. The activities of both citrate synthase (CS) and isocitrate dehydrogenase were significantly decreased in the presence of 0.9 g/l NH4Cl (e.g., 40% and 66%, respectively, in cells incubated for 95 h), while ammonium assimilation occurred via the glutamate dehydrogenase reaction. Furthermore, growth limitation occurred even in the presence of 0.5 g/l NH4Cl, when the DO concentration decreased from 135 to 30 microM. The activities of both CS and succinate dehydrogenase were dramatically decreased in cells grown at the lower DO concentration (e.g., 90% and 93% respectively, in a 95 h incubation), while ammonium assimilation was limited due to the low activities of both glutamate dehydrogenase and glutamate synthase. It is concluded that the threshold of ammonium concentration at which growth of A. lipoferum is limited, depends on the DO concentration in the medium.