Thepur3gene from thepurcluster encodes a monophosphatase essential for puromycin biosynthesis inStreptomyces

The pur3 gene of the puromycin (pur) cluster from Streptomyces alboniger is essential for the biosynthesis of this antibiotic. Cell extracts from Streptomyces lividans containing pur3 had monophosphatase activity versus a variety of mononucleotides including 3′-amino-3′-dAMP (3′-N-3′-dAMP), (N6,N6)-dimethyl-3′-amino-3′-dAMP (PAN-5′-P) and AMP. This is in accordance with the high similarity of this protein to inositol monophosphatases from different sources. Pur3 was expressed in Escherichia coli as a recombinant protein and purified to apparent homogeneity. Similar to the intact protein in S. lividans, this recombinant enzyme dephosphorylated a wide variety of substrates for which the lowest Km values were obtained for the putative intermediates of the puromycin biosynthetic pathway 3′-N-3′-dAMP (Km=1.37mM) and PAN-5′-P (Km=1.40mM). The identification of this activity has allowed the revision of a previous proposal for the puromycin biosynthetic pathway.