The pur7 Gene from the Puromycin Biosynthetic pur Cluster of Streptomyces alboniger Encodes a Nudix Hydrolase

Abstract

ABSTRACTPur7 is the product of a gene from the puromycin biosyntheticpurcluster ofStreptomyces alboniger. It was expressed inEscherichia colias a recombinant 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 other nudix hydrolases, Pur7 has an alkaline pH optimum and a requirement for 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. TheKmandVmaxvalues for these dATP and dTTP derivatives were 120 μM and 17 μM/min and 3.45 mM and 12.5 μM/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.