Biosynthesis of Streptomycin. I. Origin of the Guanidine Group.

Although the antibiotic streptomycin was isolated and characterized a number of years ago, little knowledge has accumulated concerning its synthesis by Xtreptonzyces griseus. In I954 Numerof, Gordon, Virgona, and O’Brien (1) administered glycine and acetate labeled in either the carboxyl or the (Y carbon to cultures of X. griseus. A low extent of incorporation of 1% into the streptomycin synthesized was observed in each case. The incorporated r4C was found to be predominantly concentrat,ed in the guanidine carbons. Considerable quantities of the Y-glycine and -acetate were degraded by the organism to ‘CO,. During the same year, Hunter, Herbert, and Hockenhull (2) grew S. griseus in a r4COa atmosphere and found the streptomycin produced to be labeled with 14C exclusively in the guanidine side chains. This finding suggested that incorporation of the carbon atoms of acetate and glycine might proceed via a pathway involving COZ as an intermediate (3). Addition of L-arginine to t,he medium was observed t’o depress r4C02 incorporation into the antibiotic, thus indicating a probable role for this amino acid in the synthesis of streptomycin guanidine groups from 14COs, possibly by transamidination. In subsequent experiments, Hunter and Hockenhull (3), using uniformly labeled n-glucose-14C, found that glucose was a potent precursor of the carbons of all three moieties of streptomycin-streptidine, streptose, and N-methyl-r-glucosamine-but interestingly, practically no radioactivity was detected in the guanidine carbons. Additional evidence supporting the possibility that the guanidine side chains of streptomycin may arise by an amidine transfer was obtained in 1958 by Walker (4), who discovered that S. griseus possessed transamidinase. Finally, nutritional studies by several investigators have indicated a possible role for arginine in the biosynthesis of the antibiotic. Enhancement of streptomycin production was noted when arginine was added to cultures of the organism. Recently, Majumdar and Kutzner (5) extensively studied this stimulatory effect of arginine. The results obtained by other investigators are summarized in their report. In the present study, direct evidence is presented that the guanidine carbons of streptomycin arise by transamidination. Administration of L-arginine-guanidino-14C to S. griseus cultures resulted in marked incorporation of isotope into the guanidine side chains. Bicarbonate-14C administered under similar conditions displayed little incorporation. Furthermore, a correla-