RESUMO
A novel, cold-active and highly alkaliphilic esterase was isolated from an Antarctic desert soil metagenomic library by functional screening. The 1,044 bp gene sequence contained several conserved regions common to lipases/esterases, but lacked clear classification based on sequence analysis alone. Moderate (<40%) amino acid sequence similarity to known esterases was apparent (the closest neighbour being a hypothetical protein from Chitinophaga pinensis), despite phylogenetic distance to many of the lipolytic "families". The enzyme functionally demonstrated activity towards shorter chain p-nitrophenyl esters with the optimal activity recorded towards p-nitrophenyl propionate (C3). The enzyme possessed an apparent T(opt) at 20°C and a pH optimum at pH 11. Esterases possessing such extreme alkaliphily are rare and so this enzyme represents an intriguing novel locus in protein sequence space. A metagenomic approach has been shown, in this case, to yield an enzyme with quite different sequential/structural properties to known lipases. It serves as an excellent candidate for analysis of the molecular mechanisms responsible for both cold and alkaline activity and novel structure-function relationships of esterase activity.
Assuntos
Temperatura Baixa , Esterases/isolamento & purificação , Microbiologia do Solo , Sequência de Aminoácidos , Regiões Antárticas , Sequência de Bases , Primers do DNA , Esterases/química , Esterases/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Metagenômica , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
A novel esterase was identified through functional screening of a metagenomic library in Escherichia coli obtained from Antarctic desert soil. The 297-amino-acid sequence had only low (<29%) similarity to a putative esterase from Burkholderia xenovorans. The enzyme was active over a temperature range of 7 to 54 degrees C and at alkaline pH levels and is a potential candidate for industrial application.
Assuntos
DNA/genética , DNA/isolamento & purificação , Esterases/genética , Microbiologia do Solo , Regiões Antárticas , Burkholderia/genética , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
The aerobic archaea possess four closely spaced, adjacent genes that encode proteins showing significant sequence identities with the bacterial and eukaryal components comprising the 2-oxoacid dehydrogenase multi-enzyme complexes. However, catalytic activities of such complexes have never been detected in the archaea, although 2-oxoacid ferredoxin oxidoreductases that catalyze the equivalent metabolic reactions are present. In the current paper, we clone and express the four genes from the thermophilic archaeon, Thermoplasma acidophilum, and demonstrate that the recombinant enzymes are active and assemble into a large (M(r) = 5 x 10(6)) multi-enzyme complex. The post-translational incorporation of lipoic acid into the transacylase component of the complex is demonstrated, as is the assembly of this enzyme into a 24-mer core to which the other components bind to give the functional multi-enzyme system. This assembled complex is shown to catalyze the oxidative decarboxylation of branched-chain 2-oxoacids and pyruvate to their corresponding acyl-CoA derivatives. Our data constitute the first proof that the archaea possess a functional 2-oxoacid dehydrogenase complex.
Assuntos
Proteínas Arqueais/metabolismo , Cetona Oxirredutases/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas Recombinantes/metabolismo , Thermoplasma/enzimologia , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/genética , Cromatografia em Gel , Clonagem Molecular , Biologia Computacional , Cetona Oxirredutases/química , Cetona Oxirredutases/genética , Cinética , Espectrometria de Massas , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Homologia de Sequência de AminoácidosRESUMO
Those aerobic archaea whose genomes have been sequenced possess a single 4-gene operon that, by sequence comparisons with Bacteria and Eukarya, appears to encode the three component enzymes of a 2-oxoacid dehydrogenase multienzyme complex. However, no catalytic activity of any such complex has ever been detected in the Archaea. In the current paper, we have cloned and expressed the first two genes of this operon from the thermophilic archaeon, Thermoplasma acidophilum. We demonstrate that the protein products form an alpha2beta2 hetero-tetramer possessing the decarboxylase catalytic activity characteristic of the first component enzyme of a branched-chain 2-oxoacid dehydrogenase multienzyme complex. This represents the first report of the catalytic function of these putative archaeal multienzyme complexes.
