RESUMO
Computational analysis predicted three potential hydrophobic transmembrane alpha-helices within the Ura2 multidomain protein of Saccharomyces cerevisiae, the C-terminal subdomain of which catalyses the second step of uridine-monophosphate biosynthesis by its L-aspartate carbamoyltransferase activity (EC 2.1.3.2). The fourth step of pyrimidine biosynthesis is catalysed by dihydro-orotate dehydrogenase (Ura1 protein; EC 1.3.99.11), which was similarly characterized as a peripheral membrane protein. Ex situ, the activities of the investigated enzymes were associated both with isolated yeast membranes, fractionated by differential centrifugation to remove intact nuclei, and with soluble cytoplasmic proteins.
Assuntos
Aspartato Carbamoiltransferase/metabolismo , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , Complexos Multienzimáticos/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Oxirredutases/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Aspartato Carbamoiltransferase/biossíntese , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/biossíntese , Membrana Celular/enzimologia , Citoplasma/enzimologia , Di-Hidro-Orotato Desidrogenase , Corantes Fluorescentes/química , Indóis/química , Dados de Sequência Molecular , Complexos Multienzimáticos/biossíntese , Oxirredutases/biossíntese , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/fisiologiaRESUMO
A 4.2-kb SphI-BamHI fragment of chromosomal DNA from Streptomyces granaticolor was cloned and shown to encode a protein with significant sequence similarity to the eukaryotic protein serine/threonine kinases. It consists of 701 amino acids and in the N-terminal part contains all conserved catalytic domains of protein kinases. The C-terminal domain of Pkg2 contains seven tandem repeats of 11 or 12 amino acids with similarity to the tryptophan-docking motif known to stabilize a symmetrical three-dimensional structure called a propeller structure. The pkg2 gene was overexpressed in Escherichia coli, and the gene product (Pkg2) has been found to be autophosphorylated at serine and threonine residues. The N- and C-terminal parts of Pkg2 are separated with a hydrophobic stretch of 21 amino acids which translocated a PhoA fusion protein into the periplasm. Thus, Pkg2 is the first transmembrane protein serine/threonine kinase described for streptomycetes. Replacement of the pkg2 gene by the spectinomycin resistance gene resulted in changes in the morphology of aerial hyphae.
Assuntos
Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Streptomyces/enzimologia , Streptomyces/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , DNA Bacteriano/genética , Escherichia coli/genética , Expressão Gênica , Genes Bacterianos , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Mutação , Fenótipo , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Streptomyces/ultraestruturaRESUMO
The structural genes, pkg4 and pkg3, encoding two putative protein serine/threonine kinases in Streptomyces granaticolor, have been cloned and sequenced. The genes were isolated after screening genomic sublibraries with specific probes obtained by PCR amplification of chromosomal DNA using degenerate primers which correspond to amino acid sequences highly conserved in eukaryotic protein Ser/Thr kinases. The sequences of these genes predict polypeptide chains of 761 and 780 amino acids for Pkg4 and Pkg3, respectively. The genes are separated by only 2 bp and therefore probably constitute an operon. pkg4, which is positioned upstream of pkg3, contains a UUALeu codon suggesting a developmental-dependent mode of expression. The amino-terminal half of both proteins clearly shares similarities with the family of protein Ser/Thr kinases. Both proteins studied also possess a region rich in Pro and Ala residues and a repeating motif of 11 amino acid residues, the function of which is unknown, in the carboxy-terminal domain. Expression of pkg4 in Escherichia coli gave rise to two different forms: a soluble protein autophosphorylated at threonine residues and an insoluble form phosphorylated at threonine and serine residues. In contrast, when pkg3 was expressed in E. coli, no autophosphorylation was detected either in vivo or in vitro.
