title: The pur3 gene from the pur cluster encodes a monophosphatase essential for puromycin biosynthesis in Streptomyces.
abstract: 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 = 1.37 mM) and PAN-5'-P (Km = 1.40 mM). The identification of this activity has allowed the revision of a previous proposal for the puromycin biosynthetic pathway.
authors.affiliation: Centro de Biología Molecular Severo Ochoa, Departamento de Biología Molecular (CSIC-UAM), Universidad Autónoma Madrid, Cantoblanco, 28049 Madrid, Spain.
authors.forename: María Blanca
authors.initials: MB
authors.lastname: Sánchez
authors.forename: Patricia
authors.initials: P
authors.lastname: Barrado
authors.forename: Antonio
authors.initials: A
authors.lastname: Jiménez
authors.forename: María
authors.initials: M
authors.lastname: Fernández Lobato
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D005810:Multigene Family; D009711:Nucleotides / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics*; D011691:Puromycin / Q000096:biosynthesis*; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*; D048170:Streptomyces lividans; D013379:Substrate Specificity
chemical_list: D009711:Nucleotides; D011691:Puromycin; D010744:Phosphoric Monoester Hydrolases