ANTMCROBAL AGENTS AND CHEMOTHERAPY, Aug. 1976, p. 363-369 Copyright 1976 American Society for Microbiology Vol. 10, No. 2 Printed in U.S.A. Formation of Methylated and Phosphorylated Metabolites During the Fermentation Process of Verdamicin B. K. LEE,* R. G. CONDON, G. H. WAGMAN, AND M. J. WENSTEN Microbiological Sciences, Schering Corporation, Bloomfield, New Jersey 07003 Received for publication 19 April 1976 n an attempt to understand the biosynthetic processes leading to the formation of verdamicin (end product), we have examined the patterns of the formation of methylated and phosphorylated metabolites, which resulted from either the addition of x-[methyl-'4c]methionine or [32P]KH2PO4 to the fermentation. ncorporation of label from L-[methyl-'4C]methionine into the bioactive sisomicin, verdamicin, and the chromatographically polar components increased with the progression of time. Two methylated bioinactive metabolites were found in the culture broth after removal of the methylated bioactive metabolites. n contrast to the bioactive metabolites, incorporation of the methyl-'4c label into the two methylated bioinactive metabolites decreased with the progression of time. A phosphorylated bioinactive metabolite (nonmethylated) was also found in the culture broth, fermented in the presence of [32P]KH2PO4. The role of the phosphorylated metabolite in the biosynthesis of the bioactive metabolites cannot yet be explained. The origins of the methyl groups of gentamicins and sisomicin have been studied and reported (2-4; B. K. Lee, R. G. Condon, G. H. Wagman, M. J. Weinstein, and E. Katz, manuscript in preparation). After recent findings in our laboratories that L-[methyl-14C]methionine is an excellent methyl donor to verdamicin, as it is for the gentamicins and sisomicin, the patterns of the formation of both bioactive and nonbioactive methylated metabolites during the verdamicin fermentation processes were examined. The accumulation of a phosphorylated streptomycin in the culture broth of Streptomyces griseus is known (5-7), and the subsequent hydrolysis of the phosphorylated antibiotic to streptomycin by an enzyme, produced in the cells of the same organism, has been reported (11). Uridine-5'-pyrophosphate derivatives were isolated from a penicillin-treated culture broth ofstaphylococcus aureus (8-10). The verdamicin-producing Micromonospora grisea strain has been fermented in the presence of [32P]KH2PO4, and a crude 32P-labeled metabolite has been isolated from the spent portion of the fermentation broth. or 1.9,uCi of '4C-labeled "B" fraction (as described in the following section) was added at 24 h after inoculation. Fractionation of metabolites. After harvest of the fermentation broth (50 ml each in 300-ml flasks) at various time intervals (days 3, 5, and 7), 0.5 g of oxalic acid was added, and the ph of the broth was brought to 2.0 with 12 N H2SO4. The acidified fermentation broth was shaken for 1 h at 280C and neutralized with concentrated NH40H. The neutralized broth was centrifuged, and 1.5 g of RC-50 resin (Rohm and Haas) in NH4+ form was added to the supernatant. The mixture (resin and supernatant) was shaken for 1 h at 28 C, the solution was decanted into another flask, and the resin was washed three times with distilled water. The washed resin was eluted twice with 15-ml portions of 2 N NH40H, and the combined eluates were concentrated to dryness to obtain fraction designated "A." To the spent portion, which was decanted into another flask after the adsorption of the A fraction on the RC-50 resin, 1.5 g of R-120 resin (Rohm and Haas) in Na+ form was added, and the mixture (resin and the spent portion) was shaken for 1 h at 28 C. The solution was decanted, and the resin was washed three times with distilled water. The washed resin was eluted twice with 15-ml portions of 2 N NH40H, and the combined eluates were concentrated to dryness to obtain the designated "B" fraction. Bioassay and radioactivity assay. Previously de- MATERALS AND METHODS scribed procedures (4, 12) were used. Strain. M. grisea, NRRL 3800, was used throughout the investigation. and the B fractions were developed in a descending Paper chromatography. Chromatograms of the A Fermentation. Procedures similar to those described in reference 12 were used, except that 30 gci 17% ammonia (2:1:1 by volume) and in an ascending system of the lower phase of chloroform-methanolof L-[methyl-14C]methionine, 24,uCi of [32P]KH2PO4, system of n-propanol-pyridine-acetic acid-water 363
364 LEE ET AL. (15:10:3:12), respectively, on Whatman no. 1 filter paper. RESULTS Methylated metabolites in A fraction. After addition of L-[methyl-'4C]methionine at 24 h, a time course study of incorporation of label into the methylated metabolites showed a progressive increase of the incorporation into the A fraction, in contrast to that into the B fraction (see the section, Fractionation of metabolites, in Materials and Methods; Table 1). Five radioactivity peaks representing (i) the polar components, (ii) a bioinactive component (A2), which migrated between the polar components and sisomicin, (iii) sisomicin, (iv) verdamicin, and (v) a least polar bioinactive component (A1) were observed (Table 2, Fig. 1 and 2). Radioactivity incorporated into all of the five peaks increased in parallel with the progression of time. The early (3-day) sample showed only three radioactivity peaks, representing the polar components, the A2 component, and sisomicin. The other two peaks, representing verdamicin and the Al component, showed up in the late samples (Fig. 2). Methylated metabolites in B fraction. There were at least five ninhydrin-positive bioinactive metabolites present in the B fraction, and two (components 2 and 5) of the five were methylated metabolites (Fig. 3). The weight of the B fraction was approximately ANTMCROB. AGENTS CHEMOTHER. seven to eight times greater than that of the A fraction in all cases. Of 30,uCi of radioactive methionine added at 24 h, 3.83,uCi was incorporated into the B fraction of the 3-day sample. This represents a 32-fold ratio of the radioactivity as compared with that found in the A fraction. The ratio of the radioactivity incorporated, respectively, into the B and A fractions decreased from 32:1 in day 3, to 6:1 in day 5, and to 4:1 in day 7 (Table 2, Fig. 2 and 3). n other words, both methylated components 2 and 5 in the B fraction showed the highest radioactivity in the early stage of the fermentation, but lost radioactivity gradually as the fermentation progressed further. The loss of the radioactivity was much greater in the case ofcomponent 2, as compared with component 5 (Table 1, Fig. 3). Phosphorylated metabolite in B fraction. Of 24 /.Ci of [32P]KH2PO4 added at 24 h, 0.08 /Xi was found to be incorporated into the B fraction of a 7-day sample. Out of the five ninhydrin-positive metabolites present in the B fraction, only one radioactive component (B. K. Lee et al., manuscript in preparation) was detectable (Fig. 4). Thus, component 4 was phosphorylated and ninhydrin positive, but a nonmethylated metabolite. Bioconversion of B fraction to A fraction. When a verdamicin fermentation was conducted in the presence of methyl-'4c-labeled B fraction (28.9 mg, containing 1.9,.Ci ofradioactivity) added at 24 h, radioactivity incorporated Duration of TABLE 1. Fraction A Fractionation of metabolites Fraction B tion" tion" (days) days) Wt t (mg) Bioactivity Radioactivity Wt t(m) (mg) Bioactivity Radioactivity (mg) ni %b (jig) nci 3 8.1 0.18 120 0.4 58.3 0 3,830 12.8 5 7.9 1.82 390 1.3 66.9 0 2,200 7.4 7 8.0 2.04 510 1.7 61.2 0 2,020 6.7 "Fermentation was conducted in 50 ml of medium in a 300-ml flask containing 30,uCi of L-[methyl- '4C]methionine added at 24 h. b Percentage of radioactivity incorporated. TABLE 2. Radioactivity incorporated into the components of the A and the B fractions Fraction A Fraction B Duration f A2 of(days compo- Polar 1componen VerdamiCin compomentation (days) Sisomicin nent nents Component 2 Component 5 nci 1 nci % nci % nci % nci % nci % 3 0 0 0 0 10 9.2 3 2.8 95 88.0 2,220 58.0 1,610 42.0 5 19 5.0 0 0 50 13.2 11 2.9 300 79.0 740 33.6 1,460 66.4 7 45 8.5 46 8.7 120 22.7± 17 3.2 300 56.8 670 33.2 1,350 66.8 Percentage of radioactivity distribution.
VOL. 10, 1976 DAY_LAR FERMENTATON PROCESS OF VERDAMCN 365 po L 3R, AMa N. FG. 1. Bioautogram of A fractions, developed in a descending system of the lower phase of chloroform- paper. Top to bottom, Verdamicin, sisomicin, methanol-i17% ammonia (2:1:1, by volume) on Whatman no. polar components. Left to right, 3-5- and 7-day samples. into the A fraction and the residual radioactivity in the B fraction were 0.024 /Gi and 0.9,uCi, respectively. Sixty-seven and thirty-three percent of the radioactivity in the A fraction were distributed into the polar components and the A2 component, respectively (Fig. 5). DSCUSSON At least five methylated ninhydrin-positive components were seen in the A fraction. Two methylated ninhydrin-positive components, and a phosphorylated but nonmethylated ninhydrin-positive component, were found in the B fraction. Among these components, only the mixture of the polar components, sisomicin, and verdamicin of the A fraction were bioactive. n contrast with the sisomicin fermentation, no bioactive components, corresponding to the two isomers of 4'-C-desmethyl-sisomicin (1), were detectable. n the early stage of fermentation, high incorporation of label from L-[methyl-'4C]methionine into components 2 and 5 of the B fraction was observed. The radioactivity incorporated into these two components decreased with the progression of time, and the decreasing rate in component 2 was much greater than in component 5. When the isolated methyl- 14C-labeled fraction B was added to a verdamicin fermentation broth, 1% of the label in the B fraction was incorporated into the polar components and the A2 component of the A fraction. Presumably, these results suggest that the methylated components 2 and 5 of the B fraction may be utilized as precursors for the polar components and the A2 component of the A fraction. n the early stage of the fermentation, the radioactive polar components and traces of the radioactive A2 component and sisomicin appeared in the A fraction. During fermentation, a trace amount of the radioactive Al component was detectable in addition to those three (previously noted) radioactive components. At the end of the fermentation, a substantial amount of radioactive verdamicin appeared, and radioactivity, incorporated into the five methylated components of the A fraction, was detected in the increasing order: the polar com-
366 LEE ET AL. ANTMCROB. AGENTS CHEMOTHER. DAY 3 _DAY DAY 5 ; -,,".1 ".N..%.. -"r -."...,.", '.. DAY 7 t f:,,. i. it.11 -.,- POLAR A2 Al SSOMCN VERDA MCN FG. 2. Radioactivity scans ofa fractions developed in chloroform-methanol-ammonia. Top to bottom, 3-, 5-, and 7-day samples. Left to right, Polar components, A2 component, sisomicin, verdamicin, and the Al component. FG. 3. Radioactivity scans of ninhydrin-treated B fractions developed in an ascending system of n- propanol-pyridine-acetic acid-water (15:10:3:12, by volume) on Whatman no. 1 paper. Top to bottom, Respective radioactivity scans and the ninhydrin-treated metabolites of the B fractions of 3-, 5-, and 7-day samples. Left to right, Components 5 (radioactive), 4, 3, 2 (radioactive), and 1.
DAY 3 i~~~~~~~~~~~~....... DAY 5..1 DAY 7 5 4 3 2 1
DAY 7 i i A.,ri,, J ~~ j ; i½j ;>t!^ 1>/SQlv fta.t h\, / 5 4 3 2 1 FG. 4. Radioactivity scan of a ninhydrin-treated B fraction of a 7-day sample, developed in propanolpyridine-acetic acid-water. Left to right, Ninhydrin-positive components 5, 4 (32P labeled), 3, 2, and 1. / POLAR 1A2 SSOMCN VERDAMCN FG. 5. Bioautogram and radioactivity scan of the A fr-action derived from the verdamicin fermentation conducted in the presence of the added methyl-w4c-labeled B fr-action. The papergram was developed in chloroform-methanol-ammonia. Left to right of the top, Polar components, sisomicin, and verdamicin. Left to right of the bottom, Radioactive peaks corresponding to the polar components and the 1A2 component. Note that no radioactive peaks representing sisomicin or verdamicin were found. 368
VOL. 10, 1976 ponents > sisomicin > verdamicin > the Al component > the A2 component. At present, little is known about how these different methylated metabolites are involved in the biosynthesis of verdamicin. The phosphorylated metabolite (component 4) present in the B fraction has not yet been characterized. Characterization of this metabolite will be very useful in learning the roles played by the substance on the biosynthesis of verdamicin. ACKNOWLEDGMENTS We wish to express gratitude to J. A. Marquez and R. Jaret for their criticisms and suggestions, and to D. Buffardi for typing the manuscript. LTERATURE CTED 1. Davis, D. H., D. Greeves, A. K. Mallams, J. B. Morton, and R. W. Tkach. 1975. Structures of the aminoglycoside antibiotics 66-40B and 66-40D produced by Micromonospora inyoensis. J. Chem. Soc. Perkin Trans. 1:814-818. 2. Lee, B. K., R. G. Condon, G. H. Wagman, and E. Katz. 1975. Micromonospora-produced gentamicin components. Antimicrob. Agents Chemother. 9:151-159. 3. Lee, B. K., R. G. Condon, G. H. Wagman, K. Byrne, and C. Schaffner. 1974. ncorporation of L-methionine-methyl-'4C into gentamicins.. Large-scale preparation of methyl-'4c-gentamicins. J. Antibiot. FERMENTATON PROCESS OF VERDAMCN 369 27:822-825. 4. Lee, B. K., R. T. Testa, G. H. Wagman, C. M. Liu, L. McDaniel, and C. P. Schaffner. 1973. ncorporation of L-methionine-methyl-'4C into gentamicins. J. Antibiot. 26:728-731. 5. Miller, A. L., and J. B. Walker. 1970. Accumulation of streptomycin-phosphate in cultures of streptomycin producers grown on a high phosphate medium. J. Bacteriol. 104:8-12. 6. Nomi, R., and 0. Nimi. 1959. Biosynthesis of streptomycin. V. Chemical structure of a streptomycin precursor. Agric. Biol. Chem. 33:1459-1463. 7. Nomi, R., 0. Nimi, T. Miyazaki, A. Matsuo, and H. Kiyohara. 1966. Biosynthesis of streptomycin.. solation of a natural precursor and an enzymatic substance transforming the precursor to streptomycin. Agric. Biol. Chem. 30:1269-1276. 8. Park, J. T. 1952. Uridine-5'-pyrophosphate derivatives.. solation from Staphylococcus aureus. J. Biol. Chem. 194:877-884. 9. Park, J. T. 1952. Uridine-5'-pyrophosphate derivatives.. A structure common to three derivatives. J. Biol. Chem. 194:885-895. 10. Park, J. T. 1952. Uridine-5'-pyrophosphate derivatives.. Amino acid-containing derivatives. J. Biol. Chem. 194:897-904. 11. Walker, J. B., and M. Skorvago. 1973. Phosphorylation of streptomycin and dihydrostreptomycin by Streptomyces. J. Biol. Chem. 218:2435-2440. 12. Weinstein, M. J., G. H. Wagman, J. A. Marquez, R. T. Testa, and J. A. Waitz. 1975. Verdamicin, a new broad spectrum aminoglycoside antibiotic. Antimicrob. Agents Chemother. 7:246-249.