RESUMO
The gene mak1FN coding for maltokinase from Actinoplanes missouriensis is located in a cluster similar to glycogen metabolism clusters identified in Streptomyces coelicolor. Sequence comparisons demonstrate that mak1-related genes coding for homologous proteins are present in many bacterial genomes including taxonomic distantly related groups such as Rhodospirillales or green sulfur bacteria. More than 50% of the aligned sequences are longer than the mak1 gene from A. missouriensis, and the N-terminal portion of these putative maltokinases exhibit high sequence homologies with trehalose synthases. A more detailed sequence comparison indicates a relationship of maltokinases to aminoglycoside phospho-transferases and protein kinases. Transformation of S. lividans with plasmid vectors containing either the mak1 gene from A. missouriensis or the pep2 gene from S. coelicolor resulted in recombinant strains, which produced measurable amounts of maltokinase activity. The proteins Pep2 and Mak1 were over expressed with Streptomyces lividans 66 as a heterologous host and further characterized. The possible physiological function of maltokinases is discussed.
Assuntos
Micromonosporaceae/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Streptomyces coelicolor/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Sequência Conservada , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , Estabilidade Enzimática , Expressão Gênica , Genes Bacterianos , Glucosiltransferases/genética , Concentração de Íons de Hidrogênio , Canamicina Quinase/genética , Micromonosporaceae/genética , Dados de Sequência Molecular , Família Multigênica , Fosfotransferases (Aceptor do Grupo Álcool)/isolamento & purificação , Filogenia , Proteínas Quinases/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Rhodospirillales/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência , Streptomyces coelicolor/genética , Streptomyces lividans/genética , Streptomyces lividans/metabolismo , TemperaturaRESUMO
The first genome of a virus infecting a representative of the eubacterial genus Actinoplanes is presented. Phage phiAsp2 has a circularly permutated chromosome that consists of 58,638 bp; its G/C-bias of 70.39% resembles the hosts G + C-content (71-73% within the genus). A total of 76 open reading frames (orfs) were identified, the majority of which (63) displaying equal transcriptional orientations. Functional gene clustering is obvious as orfs coding for head and tail proteins are located close to the center in the first half of the genome and putative DNA-modifying enzymes are encoded by centrally located genes; DNA repair and recombination functions are situated in the remaining part of the genome, adjacent to a small gene cluster, the predicted proteins of which are involved in DNA packaging. Close to the left terminus there are two small regions (approximately 4.5 kb each, separated by 2.8 kb) which are homologous to the recently sequenced mycobacteriophage rosebush, however, the unique overall structure of the phiAsp2-genome does not bear resemblance to any other known viral genome. The nucleotide sequence was deposited in GenBank with the accession no. AY576796.
Assuntos
Actinomycetales/virologia , Bacteriófagos/genética , Genoma Viral , Acarbose/metabolismo , Sequência de Aminoácidos , Dados de Sequência Molecular , Fases de Leitura Aberta , Análise de Sequência de DNA , Proteínas Virais/genéticaRESUMO
Crude extracts of Actinoplanes missouriensis and related strains catalyze the ATP-dependent phosphorylation of maltose to maltose 1-phosphate. The enzyme of A. missouriensis responsible for this reaction was purified and characterized. This protein has an estimated molecular mass of 57 kDa and it is most likely a monomer. The K(m) value was 2.6 mM for maltose and 0.54 mM for ATP. Only maltose acted effectively as phosphoryl-group acceptor, and ATP was not replaceable as phosphoryl-group donor. Tryptic peptides of the enzyme were sequenced, and the sequences of these peptides will allow construction of degenerate primers to identify the gene coding for this unique kinase.
