Nucleoside Intermediates in Blasticidin S Biosynthesis Identified by the in Vivo Use of Enzyme Inhibitors

Nucleoside intermediates in blasticidin S biosynthesis identified by the in vivo use of enzyme inhibitors

STEVENJ. GOULD' Departments of Chemistry and of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331-4003, U.S.A.

JINCAN GUOAND ANJAGEITMANN Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331-4003, U.S.A.

KARL DFXESUS Department of Chemistry, Oregon State University, Corvallis, OR 97331-4003, U.S.A. Received March 9, 1993 This paper is dedicated to Professors Ian Sperzser and David B. MacLean

STEVENJ. GOULD, JINCAN GUO,ANJA GEITMANN,and KARLDmus. Can. J. Chem. 72,6 (1994). Intermediates in the biosynthesis of blasticidin S and its nucleoside co-metabolites were detected by altering fermentation conditions. Inhibitors of specific types of biochemical reactions that were expected to be involved in blasticidin biosynthesis were fed to Streptomyces griseochromogenes, in some cases with the inclusion of large quantities of the primary precursors of blasticidin S. The types of reactions and inhibitors used were (1) transaminase (aminooxyacetic acid and 2-methylglutamate), (2) amidotransferase (azaserine and 6-diazo-5-0x0-L-norleucine), (3) arginine biosynthesis (arginine hydroxamate), and (4) methyltransferase (ethionine). These manipulations apparently distorted the pools of precursors and (or) intermediates, and led to substantial accumulations of three known, previously minor, metabolites of S. griseochromogenes, cytosylglucuronic acid, pentopyranine C, and demethylblasticidin S, and of two new ones, pentopyranone and isoblasticidin S. New cytosyl metabolites were detected by HPLC with photodiode array detection. Fermentations to which arginine hydroxamate and cytosine had been added also produced three aberrant metabolites that were derived from pentopyranone and arginine hydroxamate.

STEVENJ. Gorno, JINCAN GUO,ANJA GEITMANNet KARL Dmus. Can. J. Chem. 72.6 (1994). En modifiant les conditions de fermentation, on a pu dttecter les intermediaires dans la biosynthkse de la blasticidine S et de ses comCtabolites nuclCosidCs. Des inhibiteurs de reactions biochimiques spkcifiques attendues dans la biosynthkse de la blasticidine ont CtC donnCes au Streptomyces griseochrornogenes, dans certains cas avec inclusion de grandes quantitks de prkcurseurs primaires de la blasticidine S. Les types de reactions et d'inhibiteurs utilisCs sont : (I) la transaminase (acide aminooxyacCtique et le 2-mCthylglutamate); (2) amidotransfLrase (azaserine et 6-diazo-5-0x0-L-norleucine); (3) biosynthkse de I'arginine (hydroxamate d'arginine) et (4) mCthyltransfCrase (Cthionine). Ces manipulations ont apparemment modifiC la repartition des prCcurseurs et (ou) des intermediaires et ont conduit B des accumulations importantes de trois mktabolites connus, mais anterieurement mineurs, du S. griseochromogenes, l'acide cytosylglucoronique, la pentopyranine C et la demethylblasticidine S, ainsi que deux nouveaux metabolites, la pentopyranone et l'isoblasticidine S. Grgce B la CLHP avec une photodiode comme ditecteur, on a pu mettre en evidence la presence de nouveaux metabolites cytosylCs. Les fermentations rCalisCes avec des sub- strats dans lesquels de l'hydroxamate d'arginine et de la cytosine avaient CtC ajoutCs ont aussi conduit B la formation de trois metabolites aberrants dCrivLs de la pentopyranone et de l'hydroxamate d'arginine. [Traduit par la redaction]

Blasticidin S, an antifungal antibiotic produced by Stretorny- Blasticidin S is composed of N-methyl-L-P-arginine, 2 (blas- ces griseochrornogenes first isolated by Takeuchi et al. in 1958, tidic acid), and an unusual nucleoside (cytosinine), 3. Seto et al. is used commercially for the control of Piricularia oryzae (rice (13) had established that cytosine, 4, D-glucose, 5, L-a-arginine, blast) (1). Chemical degradation (2-5) and X-ray diffraction 6, and methionine, 7, are the primary precursors (Scheme 1). studies (6, 7) yielded the nucleoside structure 1. Since then, a We first became interested in the biosynthesis of 1 because of number of structurally related nucleoside antibiotics have been the potential relevance of our studies (14) on the L-P-lysine unit characterized, including gougerotin (8), mildiomycin (9), of another nucleoside antibiotic, streptothricin F, to the origin of bagougeramines A and B (lo), arginomycin (1 1), and antibiotic the L-P-arginine moiety embedded in 1. Indeed, both p-amino Sch 36605 (12). acids proved to be derived by 2,3-aminomutase reactions in which an intramolecular migration of nitrogen from C-2 to C-3 and a companion migration of the 3-proR hydrogen to the 2- proR position takes place (15, 16). Feedings with [I-~HI-and [2,3,4,6,6-2~5j-~-g1ucoses,used to probe the biosynthesis of the cytosinine moiety, revealed that the carbinol hydrogens H-1, -2, and -3 were retained in 1, but that H-4 was lost. Retention of H-2 and H-3 indicated that the deoxygenations at these positions could not occur by simple dehydrations (17). Efforts to incorporate more advanced putative intermediates proved inconclusive. We now report these results, as well as '~uthorto whom correspondence may be addressed. those of a subsequent program of feeding inhibitors of specific COULD ET AL. 7 mL) and fed sterilely in two equal portions to a 200 mL production culture (synthetic medium) 48 and 72 h.after inoculation with a seed cul- H2N$+'oH HOAoH ture. After an additional 72 h, the fermentation was bioassayed (122 HO mg of 1 had been produced) and worked up as previously described to NH2 0H 6 yield 29.4 mg of pure 1. Liquid scintillation counting revealed that this \,AS material was not radioactive. ~-N-Ace@lcytosyl-2',3',4',6'-fetraber1zoylglucose,10 N-Acetylcytosine (19) (215 mg, 1.40 mmol) and Hg(CN)2 (783 mg, 3.10 mmol) were suspended in 6 mL of nitromethane in a dry 50-mL round-bottom flask. A distillation head was attached, the mixture heated, and ca. 1 mL of solvent was distilled. At this point, the heat was removed and a-1 -bromotetrabenzylglucose (20) (923 mg, 1.40 mmol) in CH2C12 (1 mL) was added dropwise from the top of the distillation head. An additional aliquot of nitromethane (1 mL) was added, and the mixture once again heated at reflux. After 4.5 h, the yellow suspension was cooled to 0°C and filtered. 'The solid was washed with cold types of biochemical reactions that were expected to be involved nitromethane, 30% aqueous KI, water, and EtOH. After standing over- in the biosynthesis of 1. The latter approach successfully led to night, a second crop could be recovered from the filtrate, and this was the identification of the roles of a number of intermediates in the washed as described above. The combined solids were recrystallized metabolic matrix that includes the biosynthesis of 1. from DMF-water to give 567 mg (77%): mp 250°C (dec.); IR: 1778, 1739, 1717 cm-I; 'H NMR (TFA-dl) 6: 8.72 (d, lH, J = 7.8 Hz), 8.02 Experimental (d,2H,J=7.6Hz).7.93(d,2H,J=7.4Hz),7.85(d,2H,J=7.6Hz), General 7.81 (d, 2H, J=7.6 Hz), 7.69-7.61 (m, 3H), 7.55 (t, lH, J=7.6 Hz), [l-2~]-~-~lucosewas obtained from Cambridge Isotope Laborato- 7.49-7.32(m, 9H), 6.74(bm, lH), 5.56(d, lH, J=9.0Hz), 6.41 (t, lH, ries. Other organic chemicals were obtained from either Aldrich or J=9.5 Hz),6.15(t, lH, J=9.6Hz),5.93(t, lH, J=9.2Hz),4.87(dd, Sigma Chemical Company. IH, J = 12.4, 1.9 Hz), 4.814.73 (m, 2H), 2.43 (s, 3H); 13c NMR (TFA-dl) 6: 178.54, 178.36, 172.63, 171.76, 171.59, 162.24, 161.54, Preparation of [1 -14~]-blastidicacid, 2a 155.83, 154.05, 149,58, 148.34, 138.25, 138.03, 137.82, 137.61, A sample of [l "-14~]-1hydrochloride (485 mg, 1.06 mmol, 5.97 x 132.58, 132.45, 131.61, 131.53, 131.36, 130.39, 129.53, 129.01, lo6 dpdmmol) was dissolved in 6 N HCl(10 mL), and stirred at room 99.57,85.96,78.29,76.60,75.24,71.96,65.64,25.93. Anal. calcd. for temperature for 6 h (these conditions caused significant decomposition C40H13N301,: C 67.52; H 1.84, N 5.9 1; found: C 67.60, H 1.70, N of 3). The mixture was neutralized to pH 4.0 and applied to an anion 5.84. exchange column (Amberlite, IRA-410, 100 mesh, OH-, 2.5 cm x 30 cm), and the initial effluent immediately loaded onto a cation exchange ~-~-~ce@lc~tos~l-2',3',4',6'-tetrabenzo~l[l-~~]~lucose,1Oa column (Amberlite, IRC-50, 50 mesh, H+, 2.5 cm x 20 cm). This was Using the same procedure, a-1 -bromotetrabenzoyl[l -2~]glucose washed first with H70 to neutrality and then with 0.5 N HC1 until the (618 mg, 0.94 mmol), N-acetylcytosine (148 mg, 0.96 mmol), and eluates no longer contained 2a. he HC1 eluate was concentrated by Hg(CN)2 (5.34 mg, 2.1 1 mmol) yielded 474 mg (66%) of 1Oa: 'H rotary evaporation at ambient temperature, and the remaining solution NMR lacked the 6: 6.56 doublet, and the 6 5.93 triplet was a doublet was lyophilized. Recrystallization of the lyophilized residue from (J = 9.5 Hz); I3cNMR 6: 85.96 (bt). EtOH gave pure 2a dihydrochloride (234 mg, 5.97 x lo6 dpdmmol, 85% yield). P-Cytosylglucose, 8 Sodium (2 mg, 0.08 mmol) in a 25-mL round-bottom flask was Preparation of [l'-14~]-cytosinine,3a washed with hexane, dried in a stream of N2, and reacted with MeOH A sample of [1'-14c]-1 hydrochloride (320 mg, 0.70 mmol, 3.85 x (2 mL) under a N2 atmosphere. 1Oa (423 mg, 0.58 mmol) was added, lo6 dpdmmol) was dissolved in 3 N H2S04 (10 mL), and heated to followed by an additional 2 mL MeOH. The suspension was stirred at 90°C for 40 h (1 8). The mixture was neutralized with BaC03 to pH 4.0, room temperature (RT) for 25 h, diluted with CH2C12(10 mL), cooled and the precipitate was removed by centrifugation at 3000 x g for 10 to O°C, and centrifuged. The resulting hygroscopic solid was dissolved min. The supernatant was applied to an anion exchange column in water and lyophilized to yield a fluffy white solid (1 13 mg, 71%) (Amberlite, IRA-410, 100 mesh, OH-, 2.5 cm x 30 cm), and the initial that had to be protected from moisture: IH NMR (D20) 6: 7.07 (d, lH, effluent discarded. The column was washed with H20 (ca. 100 mL) to J=7.6Hz),6.06(d, lH, J=7.7Hz),4.05(d, lH,J=9.1 Hz),3.90(dd, pH < 9 and then eluted with 0.5 N HC1. Fractions of ca. 20 mL were lH, J=12.5,2.0Hz), 3.77 (dd, IH, J=12.5,5.2Hz),3.72(dd, lH, J= collected and monitored for 3 by HPLC (see conditions below; tR = 3.4 9.0,9.1 Hz),3.67(t, lH, J=8.8Hz),3.65-3.61 (m, 1H),3.53(dd, lH, min). Appropriate fractions were lyophilized and the crude product J = 9.3, 9.2 Hz); 13c NMR (D20) 6: 167.53, 159.45, 143.43, 98.51, was recrystallized from H20-acetone to obtain pure 3a (69.4 mg, 3.54 84.76,80.20,77.82,72,75,70.70,62.09. Anal. calcd. for CIOH15N306: x lo6 dpdmmol, 40% yield). C 43.96, H 5.53, N 15.39; found: C 43.90, H 5.62, N 15.1 1.

Feeding [l'-lk~cytosinine,3a ~-~~fos~l[l-~~]~lucose,8a A sample of 3a (9.3 x 10' dpm) was dissolved in deionized H20(10 Using the same quantities, 10a was deprotected to yield 8a (105 mg, mL) and fed sterilely by filtering through a Gelman membrane filter 66%): 'H NMR lacked the 6 4.05 doublet and the 6 3.72 triplet was (Product No. 4192, pore size 0.2 km) into a 200 mL production culture now a doublet (J = 9.0 Hz); 13cNMR 6: 85.76 (bt). (synthetic medium) (18) 42 h after inoculation with a seed culture. After an additional 80 h, the fermentation was bioassayed (95 mg of 1 Culture maintenance, ferrnenfation, arld bioassay conditions had been produced) and worked up as previously described (16) to Standard culture maintenance, fermentation, and bioassay condi- yield 29.0 mg of pure 1. Liquid scintillation counting revealed that this tions have been described previously (16). material was not radioactive. Inhibitor/precursor feedings Feeding [~'-~~C]-blasfidicacid, 2a Inhibitors and precursors were added as sterile-filtered (pore size A sample of 2a (1.1 x lo6 dpm) was dissolved in deionized H20(10 0.2 km) aqueous solutions at 50 h after inoculation of production 8 CAN. J. CHEM. VOL. 72, 1994 broths (100 mL in 500-mL Erlenmeyer flasks). The quantities of addi- tives were cytosine 30 mg, arginine 200 mg, methionine 200 mg, aminooxyacetic acid 40 mg, 2-methylglutamate 50 mg, arginine R0GDRO hydroxamate 200 mg, 6-diazo-5-0x0-L-norleucine30 mg, azaserine &HO - - 31 mg, ethionine 50 mg. H0 D R0 BF

HPLC analysis 5a 9a,R=k For each experiment, 0.5 mL samples were taken at the times noted in the tables, centrifuged 10 min (Eppendorf microfuge), and either immediately analyzed or stored at -20°C. Twenty microlitre aliquots "Y--"" were injected for each analysis. The conditions for analytical HPLC HN / NHR~ x GY.N / (Waters Assoc. model 600E) were cI8~adial~ak~ column, 0.8 cm 10 Hg(CN)2 R' 0 cm (Waters Assoc. 4 pm packing), eluted with 95% H20, 5% CH,CN, R1O 0.15% TFA, 1.1 mLImin, with a Waters model 990+ photodiode array detector scanning the region 200-300nm. Chromatograms were ~O~,R'=~,R~=AC printed out for absorption at 275 nm. 8a. R' =R~=H Results and discussion SCHEME3 A retrobiogenetic analysis fragmented 1into 2 or P-arginine, Numerous permutations on the same types of biochemical itself, and 3. The reconstruction of 1from 2 and 3 by a mycelial reactions could be proposed for the biosynthesis of 1 from the suspension of S. griseochromogenes had been reported (1 8), but known precursors. Since each hypothetical sequence involved was not reproducible (private communication, H. Seto), and had numerous intermediates, and in view of the negative results been tested without the benefit of isotope labels. We, therefore, described above, it was clear that alternative approaches to con- produced blasticidin S samples from biosynthetic incorpora- ventional feeding experiments were needed to overcome poten- tions of [l-14c arginine and [l-14~]glucose,and hydrolyzed tial permeability problems and to avoid the synthesis of a large each to yield [I-'l 4~]-2aand [1'-14c]-3a, respectively. Each was number of putative, but incorrect, intermediates. A novel fed to cultures of S. griseochromogenes, and the blasticidin S approach was adopted that altered fermentation conditions in produced by each was isolated and purified (16). However, nei- order to block potential biosynthetic steps. This was done by ther sample was radioactive (Scheme 2) and, furthermore, much feeding inhibitors of specific types of biochemical reactions, of the radioactivity had remained in the broth at the end of the and in some cases large quantities of primary precursors were fermentations. included. These manipulations were expected to distort the biosynthetic precursor pools and force the accumulation of biosynthetic intermediates or new metabolites in the fermentation broth. Although this empirical approach lacks control over the effects an inhibitor may have at other points in the cellular metabolism, selective inhibition of secondary metabolism with little stress on primary metabolism seemed possible because in most cases the timings of bacterial growth and antibiotic production are well differentiated (21). In comparison with attempting to develop blocked mutants, this approach appeared to offer greater flexibility in selecting the inhibition points. The selection of inhibitors for these studies was based on three assumptions: (a) the 4'-amino group is introduced after the formation of the cytosine glycosidic bond, (b) the nucleoside portion is coupled to the p-amino acid portion at a relatively late stage, and (c) 6-N-methylation occurs late in the overall pathway. Attention was therefore focused on blocking three potential biosynthetic points. These points, and the specific inhibitors tested, were (a) blocking transamination with transaminase inhibitors (aminooxyacetic acid (AOAA), 11 (22-24), and 2-methylglutamate (MeGlu), 12 (25)) or amidotransferase inhibitors (azaserine, 13 (26), and 6-diazo-5-0x0-L- We next tested cytosylglucose, 8, as the first nucleoside inter- norleucine (DON), 14 (27)); (b) blocking the availability of L- mediate. To test this, [1'-~~]-8awas synthesized as shown in a-arginine with a L-a-arginine biosynthetic inhibitors (L-argin- Scheme 3. [~-~~]-~-~lucose,5a, was converted to the l-bro- ine hydroxamate (ArgH), 15 (28,29), and 12); and (c) blocking motetrabenzoate (20) 9a and coupled with N-acetylcytosine N-methylation with a methyltransferase inhibitor (L-ethionine (19) using standard H~~+catalysis (19). Methoxide then fully (Eth), 16 (30, 3 1)). deprotected 10a to yield 8a. In the undeuterated series, a 9.1 Hz To maximize both the effect and selectivity of the inhibitors, coupling between the H-1' and H-2' NMR resonances con- it was necessary to first determine the relative timing of bacte- firmed that the P anomer had been obtained. A sample of 8a rial growth and production of 1 (BS) so that inhibitors were fed (100 mg) was fed to a culture of S. griseochromogenes, but 2~ at the onset of BS production. A synthetic medium (18) was NMR analysis of the derived 1failed to show any incorporation chosen for production broths to simplify the interpretation of of deuterium (Scheme 2). the inhibitor results. It was found that the bacteria entered log GOULD ET AL. 9

:: 1 8 I I :; .m * - : : . :: : 5,... .,,1' ,..' 20 ?: e*,, '. I. .-,--.....--.,-2.2 '.' --.' .-'.,: *.------.. 19: phase right after inoculation of production broths with the seed .. - .. culture, and reached stationary phase within 84-96 h. 1was first .. $ .. .:: . detected between 50 and 60 h after inoculation, and the accumu- .*.. i \ lation typically lasted for 5 days. Therefore, inhibitors were fed . . .I :.. . :-7, . . at 50 h. Production of 1 and its related metabolities was initially . .,-...*. 6 r ...... 3-i ...... :l...j 'x ...... assayed 168 h later. HPLC conditions were developed that sep- - arated authentic samples of 1, blasticidin H, 17 (32), and pentopyranine C (PPNC), 18 (33, 34), and a photodiode array detector was used to easily identify new cytosine-containing Ad metabolites.

H2N Control I ...... CH3 OH AOAA + Cytosine 17 ------ArgH + Cytosine

$kc,~~,R~=R~=R~=H,R~.OH I I I R4 19. R' = C02H. R2 = H, R3 = R4 = OH 0.0 2.5 5.0 7.5 10.0 OH TIME, rnin

The amounts of inhibitors initially used were comparable to FIG. 1. HPLC of Streptomyces griseochromogerzes fermentations those used in the literature, and some were subsequently with and without the addition of cytosine and the enzyme inhibitors adjusted according to the information obtained during the feed- aminooxyacetic acid (AOAA), 11, or arginine hydroxarnate (ArgH), ing experiments. In several preliminary feedings of either 11 or 15. See Experimental for conditions. 15, the production of two cytosyl metabolites was stimulated as much as 200-300% over a control broth, while the production of 1 was inhibited by 50-70%. One of these metabolites had an 12, 13, and 14. Some of these inhibitors were also co-fed with HPLC retention time identical to that of authentic 18, while the the primary precursors cytosine, arginine, and methionine. Fer- other was considerably more polar. Further studies demon- mentations with each inhibitor were assayed at 12 h intervals strated that when cytosine was co-fed with either 11 or 15, for the first 4 days, and then daily for the next 3. A number of production of these two new metabolites was increased dramat- these were repeated on two or three occasions and, although the ically (Fig. 1). These were then purified and identified by NMR absolute amounts varied from one set of fermentations to spectroscopy. The less polar compound was indeed 18. The another (each set was inoculated from the same seed culture for other, which was found to bind to both anion and cation consistency), the relative levels of metabolites were similar. exchange resins, indicating an acid, was identified as cytosyl- The more significant effects of the inhibitors on CGA (19), glucuronic acid (CGA), 19. This compound had previously PPNC (IS), DeMeBS (20), and BS (I) production are presented been isolated in milligram quantities from hundreds of litres of in Tables 1-3. Several trends were apparent, and are summa- fermentation broth (35). In these latter inhibitor feedings, accu- rized as follows. mulation of a third new component was also slightly stimulated Feeding the primary precursors 4,6, and 7 (data for the latter (ca. 2-fold). Purification by HPLC provided enough material two not shown) had a stimulatory effect on the production of all for its identification by 'H NMR spectroscopy as demethylblas- four compounds assayed. Cytosine, which had been incorpo- ticidin S (DeMeBS), 20 (36). Only a slight elevation for 20 was rated almost quantitatively into 1 as a radiolabeled tracer (1 3), observed with ethionine when assayed at 168 h, although there had a much stronger effect than the other precursors. This was was also a noticeable decrease in the amount of 1. especially true for production of 19, which was increased 30- With these initial successes, a more systematic study was fold at 96 h and 71-fold at 168 h. Co-feeding the precursors developed to examine the effects of 11,15, and 16, as well as of cytosine and arginine had an additive effect, and was the best 10 CAN. J. CHEM. VOL. 72, 1994 TABLE1. The effects of cytosine and of enzyme inhibitors on the no longer noticeable, but the enhancement of 19 was even more production of S. griseochrornogenes metabolites at 96 h pronounced. These effects were most likely due both to its action as a glutamatePLP-dependent transaminase inhibitor Additive(s)" CGA (19)~ PPNC (181~ DeMeBS (20)' BS (1)' and as an inhibitor of glutamate biosynthesis; the latter effect would indirectly inhibit arginine biosynthesis. While the effects Cytosine (4) AOAA (11) of arginine hydroxamate, 15, were very similar to those of 11, it MeGlu (12) was particularly efficient, especially in the presence of added ArgH (15) cytosine, in stimulating production of 18 (ca. 17-fold at 168 h). DON (14) Although in the initial study ethionine, 16, showed hardly Azaserine (13) any effect on 20 after 168 h, assay at the earlier times now Ethionine (16) showed that 16 caused a transitory increase of 20 and decrease AOAA/MeGlu of 1. The production of 20 was highest after 96 h, and decreased ArgWMeGlu afterwards. Possibly 16 was degraded after longer periods of 'For amounts fed, see Experimental. incubation. Nevertheless, the effect of 16 was consistent with b~elativeconcentration compared to production by a control fermentation our previous proposal that 6:-N-methylation occurs at a late to which nothing had been added. stage in the pathway (16). The effects of these metabolic inhibitors and primary precur- TABLE2. The effects of enzyme inhibitors on the produc- sors were next examined in fermentations in the standard com- tion of S. griseochromogenes metabolites at 120 h plex medium (16), which normally produced much more of 1. Although higher dosages of inhibitors were needed to bring Additive(s)' (191b (18)~ (20)~ (1)' about a comparable extent of inhibition, the same patterns of inhibition and stimulation were observed on the production of all four metabolites (data not shown). By using this system, substantial quantities of 19, 18, and 20, which were originally produced only in trace amounts, could now be easily purified (as much as 1.5 g/L of CGA) from a relatively small fermentation (typically 1 L). In addition to the four compounds already identified, HPLC analysis of the crude fermentation broth obtained from the complex medium supplemented by 1.0 g/L of "For amounts fed, see Experimental. 4 and 2.0 g/L of 15 revealed several new components. Photo- b~elativeconcentration compared to production by a control diode array detection showed that at least five of those new fermentation to which nothing had been added. components had UV spectra characteristic of the cylosine gly- cosides. Large-scale fermentations under these conditions TABLE3. The effects of cytosine and of enzyme inhibitors allowed isolation of sufficient quantities of each to characterize on the production of S. griseochromogenes metabolites at them spectroscopically as 21-25 (37). 168 h

\ 4 70.6 6.4 3.5 1.6 OH 11 3.3 3.2 0.7 0.7 21,x=o 12 7.5 2.1 1.O 0.6 22,X = NOH 15 1.6 1.9 0.4 0.6 14 1.5 1.4 0.0 0.9 13 0.5 1.2 0.9 0.9 16 3.4 1.7 1.2 0.7 H2N~NH OH 11/12 4.5 2.2 0.4 0.6 15/12 4.1 12.1 0.7 0.4 24

'For amounts fed, see Experimental. b~elativeconcentration compared to production by a control fermentation to which nothing had been added. condition for accumulation of 20 (data not shown), presumably by overwhelming the pool of S-adenosyl-L-methionine. Aminooxyacetic acid, 11, substantially inhibited the production of 1 and 20 while moderately stimulating the production of 18 and 19. In contrast, the glutamine-dependent amidotransferase inhibitors 13 and 14 showed much milder effects. The effect of 11 also appeared to be quite specific, since its inhibi- tory effect on the production of 1 could not be relieved by methionine, arginine, or P-arginine. 2-Methylglutamate had effects similar to those of 11. At 96 h The in vivo use of specific enzyme inhibitors with S. griseo- its effects on 18,20, and 1 were similar, although it enhanced 19 chromogenes fermentations has led to the accumulation of four substantially more. At later time points, the inhibition of 20 was otherwise very minor metabolites, including one (21) that had GOULD ET AL. 11 not been previously detected. This has led to an intimate view 12. R. Cooper, M. Conover, and M. Patel. J. Antibiot. 41, 123 into the individual biochemical steps that lead to blasticidin S (1988). and the metabolic matrix that includes related cytosyl nucleo- 13. H. Seto, I. Yamaguchi, N. Otake, and H. Yonehara. Argic. Biol. sides. With these large accumulations now possible, 19 was Chem. 32, 1292 (1968). confirmed as the first nucleoside intermediate (38) and CGA 14. S.J. Gould and T.K. Thiruvengadam. J. Am. Chem. Soc. 103, 6752 (198 1). synthase, the first prokaryotic glucuronosyltransferase, was iso- 15. T.K. Thiruvengadam, S.J. Gould, D.J. Aberhart, and H.J. Lin. J. lated and purified (39), demethylblasticidin S was shown to be Am. Chem. Soc. 105,5470 (1983). the last intermediate in the overall pathway by its enzymatic 16. P.C. Prabhakaran, N.T. Woo, P. Yorgey, and S.J. Gould. J. Am. conversion to 1 (40), and the mechanism and stereochemistry of Chem. Soc. 110.5785 (1988). C-3' deoxygenation was established (41). 17. S.J. Gould, C.H. Tann, P.C. Prabhakaran, and L.R. Hillis. Bioorg. Nucleoside antibiotics comprise a large and structurally Chem. 16,258 (1988). diverse group of microbial metabolites (42). Considering their 18. H. Yonehara and N. Otake. Antimicrob. Agents Chemother. 855 numbers and significant biological activities, the sparsity of (1 965). biosynthetic studies is at first surprising. However, real or antic- 19. K.A. Watanabe, M.P. Kotick, and J.J. Fox. J. Org. Chem. 35,231 ipated permeability barriers have probably been significant (1970). 20. R.K. Ness, H.G. Fletcher, Jr., and C.S. Hudson. J. Am. Chem. impediments (42). Indeed, many of the most definitive studies SOC.72,2200 (1950). have focused on the appendages to the nucleoside sugar (42- 21. J.F. Martin and A.L. Demain. Microbiol. Rev. 44, 230 (1980). 44). The in vivo use of specific enzyme inhibitors, which has 22. D.P. Wallach and N.J. Crittenden. Biochem. Pharmacol. 5, 323 proved so rewarding in the present study, is likely to be effective (1961). with other such "converging" pathways, where individual sub- 23. D.G. Brunk and D. Rhodes. Plant Physiol. 87,447 (1988). pathways can be targeted. We are currently investigating the 24. A.R. Khornutov, A.G. Gabibov, E.N. Khurs, E.A. Tolosa, A.M. effects of additional inhibitors on blasticidin biosynthesis, as Shuster, E.V. Goryachenkova, and R.M. Khomutov. In Biochern- well as exploring the utility of this approach in probing further istry of Vitamin B6: Proceedings of the 7th International Con- into the biosynthesis of streptothricin F. gress on Chemical and Biological Aspects of Vitarnin B6 Catalysis. Edited by T. Korpela and P. Christen. International Acknowledgements Union of Biochemistry Symposium. Birkhauser Verlag, Basel, Boston. 1987. pp. 317-320. Dr. Y. 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