The pur7 gene from the puromycin biosynthetic pur cluster of Streptomyces alboniger encodes a nudix hydrolase.
Pur7 is the product of a gene from the puromycin biosynthetic pur cluster of Streptomyces alboniger. It was expressed in Escherichia coli as a recombinant protein fused to a His tag and then was highly purified through a Ni(2+) column. It showed a 3'-amino-3'-dATP pyrophosphohydrolase (nudix) activity which produced 3'-amino-3'-dAMP and pyrophosphate. This is consistent with the presence of a nudix box in its amino acid sequence. As observed with other nudix hydrolases, Pur7 has an alkaline pH optimum and a requirement for Mg(2+). Among a large variety of other nucleotides tested, only 3'-amino-3'-dTTP was a Pur7 substrate, although at lower reaction rates than 3'-amino-3'-dATP. These findings suggest that Pur7 has a high specificity for the 3' amino group at the ribofuranoside moiety of these two substrates. The K(m) and V(max) values for these dATP and dTTP derivatives were 120 microM and 17 microM/min and 3.45 mM and 12.5 microM/min, respectively. Since it is well known that 3'-amino-3'-dATP is a strong inhibitor of DNA-dependent RNA polymerase, whereas 3'-amino-3'-dAMP is not, Pur7 appears to be similar to other nudix enzymes in terms of being a housecleaning agent that permits puromycin biosynthesis to proceed through nontoxic intermediates. Finally, the identification of this activity has allowed a revision of the previously proposed puromycin biosynthetic pathway.
Espinosa
J C
JC
Centro de Biología Molecular "Severo Ochoa," CSIC/UAM, Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
Tercero
J A
JA
Rubio
M A
MA
Jiménez
A
A
D000123:Acetyltransferases / Q000235:genetics* / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D002874:Chromosome Mapping; D004789:Enzyme Activation / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D016296:Mutagenesis; D010641:Phenotype; D011691:Puromycin / Q000096:biosynthesis*; D011755:Pyrophosphatases / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity
D011994:Recombinant Proteins; D000249:Adenosine Monophosphate; D011691:Puromycin; D000255:Adenosine Triphosphate; D000123:Acetyltransferases; C045336:puromycin N-acetyltransferase; D011755:Pyrophosphatases; C019757:ATP pyrophosphohydrolase