Possible evolutionary relationships between streptomycin and bluensomycin biosynthetic pathways: detection of novel inositol kinase and O-carbamoyltransferase activities

Abstract

Bluensomycin (glebomycin) is an aminocyclitol antibiotic that differs structurally from dihydrostreptomycin in having bluensidine (lD-1-O-carbamoyl-3-guanidinodeoxy-scyllo-inositol) rather than streptidine (1,3-diguanidino-1,3-dideoxy-scyllo-inositol) as its aminocyclitol moiety. Extracts of the bluensomycin producer Streptomyces hygroscopicus form glebosus ATCC 14607 (S. glebosus) were found to have aminodeoxy-scylloinositol kinase activity but to lack 1D-1-guanidino-3-amino-1,3-dideoxy-scyllo-inositol kinase activity, showing for the first time that these two reactions in streptomycin producers must be catalyzed by different enzymes.S. glebosus extracts therefore possess the same five enzymes required for synthesis of guanidinodeoxy-scylloinositol from myo-inositol that are found in streptomycin producers but lack the next three of the four enzymes found in streptomycin producers that are required to synthesize the second guanidino group of streptidine-P.In place of a second guanidino group, S. glebosus extracts were found to catalyze a Mg2e-dependent carbamoylation of guanidinodeoxy-scyllo-inositol to form bluensidine, followed by a phosphorylation to form bluensidine-P. The novel carbamoyl-P:guanidinodeoxy-scyllo-inositol O-carbamoyltransferase and ATP:bluensidine phosphotransferase activities were not detected in streptomycin producers or in S. glebosus during its early rapid growth phase. Free bluensidine appears to be a normal intermediate in bluensomycin biosynthesis, in contrast to the case of streptomycin biosynthesis; in the latter, although exogenous streptidine can enter the pathway via streptidine-P, free streptidine is not an intermediate in the endogenous biosynthetic pathway. Comparison of the streptomycin and bluensomycin biosynthetic pathways provides a unique opportunity to evaluate those proposed mechanisms for the evolutionary acquisition of new biosynthetic capabilities that involve gene duplication and subsequent mutational changes in one member of the pair. In this model, there are at least five pairs of enzymes catalyzing analogous reactions that can be analyzed for homology at both the protein and DNA levels, including two putative pairs of inositol kinases detected in this study.Actinomycete soil bacteria have a large excess of DNA in their genomes over that required for growth and morphological differentiation. Much of this excess DNA codes for enzymes responsible for the unique biosynthetic abilities of actinomycetes, which as a group produce 70% of the known antibiotics plus numerous other idiolites (secondary metabolites). The mechanisms and selection procedures by which the diverse pathways for idiolite biosynthesis have evolved and are maintained are problems of both theoretical and practical interest. Model systems for addressing these problems must be amenable to both enzymatic and genetic analysis. The streptomycin biosynthetic pathway fulfills these two requirements for a model system (32).Enzymatic studies on biosynthesis of the streptidine moiety of dihydrostreptomycin (Fi...

Enzymatic Synthesis of Aminoglycoside Antibiotics: Novel Adenosylmethionine:2-Deoxystreptamine N -Methyltransferase Activities in Hygromycin B- and Spectinomycin-Producing Streptomyces spp. and Uses of the Methylated Products

“…2. Enzymatic reactions previously discovered by my laboratory to be involved in biosynthesis of the streptidine core of streptomycin (13,16,21) and the bluensidine core of bluensomycin (14,17). Enzymes U and V are absent in streptomycin producers, and enzymes H, I, and J are absent in bluensomycin producers (17).…”

“…2) (19)(20)(21)(22)(23)(24)(25). Subsequently the reactions specifically required for synthesis of the bluensidine core of bluensomycin were identified (14,17). More recently, my laboratory has initiated studies of the enzymatic synthesis of the aminocyclitol cores of spectinomycin (IX) (18) and of hygromycin B (IV).…”

Enzymatic synthesis of aminocyclitol moieties of aminoglycoside antibiotics from inositol by Streptomyces spp.: detection of glutamine-aminocyclitol aminotransferase and diaminocyclitol aminotransferase activities in a spectinomycin producer

“…of the streptidine moiety of streptomycin and hydroxystreptomycin and the bluensidine moiety of bluensomycin (glebomycin), previously discovered by our laboratory (16,(18)(19)(20)24 -amino groups of certain diaminocyclitols by the reactions of equations 9 to 12. The reactions are generalized here for streptamine synthesis as might occur in the synthesis of the SS-56C antibiotic (7) or 2-hydroxygentamicin by Daum's idiotroph (5,6).…”

“…glebosus ATCC 14607 (S. glebosus) came from the American Type Culture Collection. All three cultures were maintained on the same slants, grown on the same medium, and harvested in the early stationary phase (42 to 48 h), as described previously for S. glebosus (19), except that S. griseocarneus was harvested by centrifugation. Cell extracts of mycelia were prepared by sonication, and supernatants were stored at Ϫ20ЊC as described for S. glebosus (19).…”

“…All three cultures were maintained on the same slants, grown on the same medium, and harvested in the early stationary phase (42 to 48 h), as described previously for S. glebosus (19), except that S. griseocarneus was harvested by centrifugation. Cell extracts of mycelia were prepared by sonication, and supernatants were stored at Ϫ20ЊC as described for S. glebosus (19). myo-[U 14 C]inositol (30 Ci/ mol) from American Radiochemicals was evaporated to dry- b This value represents a 70% conversion of labeled myo-inositol to labeled aminodeoxyinositol, providing a good source of labeled aminodeoxyinositol for antibiotic research (cf.…”