Streptomycin Biosynthesis: SEPARATION AND SUBSTRATE SPECIFICITIES OF PHOSPHATASES ACTING ON GUANIDINODEOXY-SCYLLO-INOSITOL PHOSPHATE AND STREPTOMYCIN-(STREPTIDINO)PHOSPHATE

Abstract

Abstract Two distinct guanidinocyclitol phosphate phosphatase activities have been detected in extracts of streptomycin-producing strains of Streptomyces and separated on a Sephadex G-200 column. Both enzymatic activities appear following a phase of rapid mycelial growth on complex media, and the enzymes thus are idiophase enzymes. Both enzymes require Mg2+ for activity, and both are inhibited by inorganic phosphate. Guanidinodeoxy-scyllo-inositol phosphate phosphatase catalyzes an essential reaction in the biosynthesis of streptidine (1,3-diguanidino-1,3-dideoxy-scyllo-inositol), a component of streptomycin. 2-Guanidino-2-deoxy-neo-inositol phosphate and N'-amidinostreptamine phosphate can also serve as substrates. Activity is inhibited by sulfhydryl reagents and stimulated by dithiothreitol. Periodate degradation studies suggest that substrate phosphate is esterified at position 4, para to the guanidino group. Streptomycin-(streptidino)phosphate phosphatase hydrolyzes phosphate esterified with streptidine, either free or combined in streptomycin derivatives, such as dihydrostreptomycin-(streptidino)phosphate and phosphoryldihydrostreptomycin-(streptidino)phosphate. Tris competes effectively with water as an acceptor of phosphate from these compounds; O-phosphoryl-Tris, once formed, is only slowly hydrolyzed. Activity is not inhibited by sulfhydryl reagents. This enzyme also acts to a lesser extent on N-amidinostreptamine phosphate and N'-amidinostreptamine phosphate. Periodate degradation studies on the latter two compounds have suggested that the natural N isomer has its amino group para to the phosphate, whereas the unnatural N' isomer has its guanidino group para to the phosphate. A current concept of the pathway of streptidine biosynthesis is presented.