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 o f Streptomyces alboniger. It was expressed in Escherichia coli as a recombi nant protein fused to a His tag and then was highly purified through a Ni2 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 o ther nudix hydrolases, Pur7 has an alkaline pH optimum and a requirement fo r Mg2+. 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 mu M and 17 m u M/min and 3.45 mM and 12.5 mu M/min, respectively. Since it is well known that 3'-amino-3'-dATP is a strong inhibitor of DNA-dependent RNA polymeras e, whereas 3'-amino-3'-dAMP is not, Pur7 appears to be similar to other nud ix enzymes in terms of being a housecleaning agent that permits puromycin b iosynthesis to proceed through nontoxic intermediates. Finally, the identif ication of this activity has allowed a revision of the previously proposed puromycin biosynthetic pathway.