RESUMO
The genome of Bacillus cereus contains 26 Nudix hydrolase genes, second only to its closest relative, Bacillus anthracis which has 30. All 26 genes have been cloned, 25 have been expressed, and 21 produced soluble proteins suitable for analysis. Substrates for 16 of the enzymes were identified; these included ADP-ribose, diadenosine polyphosphates, sugar nucleotides, and deoxynucleoside triphosphates. One of the enzymes was a CDP-choline pyrophosphatase, the first Nudix hydrolase active on this substrate. Furthermore, as a result of this and previous work we have identified a new sub-family of the Nudix hydrolase superfamily recognizable by a specific amino acid motif outside of the Nudix box.
Assuntos
Bacillus anthracis/enzimologia , Bacillus cereus/enzimologia , Pirofosfatases/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Genes Bacterianos , Genoma Bacteriano , Cinética , Dados de Sequência Molecular , Família Multigênica , Plasmídeos/metabolismo , Ligação Proteica , Especificidade da Espécie , Difosfato de Uridina/químicaRESUMO
Gene ytkD of Bacillus subtilis, a member of the Nudix hydrolase superfamily, has been cloned and expressed in Escherichia coli. The purified protein has been characterized as a nucleoside triphosphatase active on all of the canonical ribo- and deoxyribonucleoside triphosphates. Whereas all other nucleoside triphosphatase members of the superfamily release inorganic pyrophosphate and the cognate nucleoside monophosphate, YtkD hydrolyses nucleoside triphosphates in a stepwise fashion through the diphosphate to the monophosphate, releasing two molecules of inorganic orthophosphate. Contrary to a previous report, our enzymological and genetic studies indicate that ytkD is not an orthologue of E. coli mutT.