826 THE JOURNAL OF MAY 1989

ENZYMIC INACTIVATION OF mentation has been shown to similarly convert LINCOSAMINIDE AND and .7) By manipula- ANTIBIOTICS : DIVALENT METAL tion of reaction pH and substrate, the crude CATION AND COENZYME enzymecan be employed in the investigation of SPECIFICITIES macrolide phosphorylation, lincosaminide phos- phorylation and lincosaminide nucleotidylation. V. P. Marshall, W. F. Liggett In these currently reported investigations of and J. I. Cialdella the crude enzyme, pirlimycin (Fig. 1) and (Fig. 2) were employed respectively as the Research Laboratories, The Upjohn Company, lincosaminide and macrolide test substrates. Kalamazoo, MI 49001, U.S.A. Wehave reported the coenzyme and metal (Received for publication November 21, 1988) co factor specificities, as well as the optimal times of production of the conversion enzymeactivities coelicolor Miiller, NRRL3532 by S. coelicolor Miiller. (UC 5240) in fermentation converts lincosaminide Growthof S. coelicolor and Preparation of the antibiotics to mixtures of inactive lincosaminide- Crude Enzyme 3-(5'-ribonucleotides) and lincosaminide-3-0- S. coelicolor Miiller was grown as described phosphates.1*^ As might be expected, crude in two of our previous publications,3»6) and was enzyme preparations of S. coelicolor Mtiller harvested by centrifugation in the cold at 104 x g catalyze the formation of both types of these for 15 minutes. Sedimented mycelial mass inactivation products. However, these conver- (10g) was resuspended in 10ml of 100mM sions are pH dependent with nucleotidylation potassium phosphate (pH 7.0) and was centri- (Fig. 1) occurring optimally near pH 6.03»4) fuged as described previously. The washed and phosphorylation (Fig. 1) near pH 8.5.5) cellular material was then resuspended in 10 ml The S. coelicolor Miiller crude enzyme also of 10 mMpotassium phosphate (pH 7.5), which catalyzes the inactivation of , contained EDTAat 500 /^g/ml and was lysed oleandomycin, spiramycin, leucomycin A3 and by egg-white lysozyme (Sigma) (2mg/ml of tylosin (Fig. 2) through phosphorylation at their resuspended mycelia) using the procedure of 2/-positions,6) while S. coelicolor Miiller in fer- Hey and Elbein.8) The resulting cell free ex-

Fig. 1. The structures of lincosaminide substrate and products. VOL. XLII NO. 5 THE JOURNAL OF ANTIBIOTICS 827

Fig. 2. The structure of tylosin and tylosin-2'-0-phosphate.

Tylosin R = H O Tylosin-2'-0-phosphate R=P(OH)2

tract was used as the crude enzyme. Crude 20mm when applied to seeded agar trays. enzyme protein was quantitated by the Bio-Rad Through comparison of the experimental biounit method which is based on the procedure of activities to those produced by standardized Bradford.9) The protein concentration of the pirlimycin solutions, jug values were assessed. crude enzyme averaged ca. 4mg/ml. In all Recently, crude enzyme preparations of Sta- cases, phosphate buffers were prepared by ad- phyloccocci were reported to convert justing the pH of solutions of KH2PO4with and to their ribonucleotides in the KOH. presence of Mg2+ and ATP, GTP, UTP or Enzymic Nucleotidylation of Pirlimycin CTP.10) This procedure was performed using reaction Enzymic Phosphorylation of Pirlimycin volumes of 20 ml. Twenty-five-ml Erlenmeyer The reaction conditions were identical to those flasks were employed as the reaction vessels. reported above for pirlimycin nucleotidyla- The reaction mixtures contained nucleoside-5'- tion with the following exceptions. Tris buffer triphosphates (pH 7.0) (Sigma) 50 /zmol, crude (pH 8.0) was added at a concentration of 5 ^mol/ enzyme protein 400 ^g, divalent metal cations ml of reaction volume and the reaction pH was 40 it*mol, pirlimycin-HC1 (Upjohn) 223 ^g and adjusted to pH 8.5 with KOH.Under these potassium phosphate (pH 7.0) 5 ^mol added per reaction conditions, lincosaminides are known ml of distilled water. The reaction mixtures to be converted to lincosaminide-3-O-phos- were adjusted to pH 6.0 with HC1and the reac- phates.5) Pirlimycin quantitation was performed tions were stirred with small magnetic bars at as described above. Tris buffer was prepared 25°C for the designated periods of time. by adjusting the pHof solutions of Tris base Using the reaction conditions described, pirli- mycin and other lincosaminides are knownto be with HC1. inactivated through conversion to lincosaminide- Enzymic Phosphorylation of Tylosin 3-(5'-ribonucleotides).3>4) Therefore, inactiva- Enzymic phosphorylation of tylosin was per- tion under these conditions is known to be formed using a reaction volume of 20ml. equivalent to nucleotidylation. A biounit assay Twenty-five-ml Erlenmeyer flasks were employed employing Micrococcus luteus UC130 was used as the reaction vessels. The reaction mixtures to quantitate these inactivations. One biounit contained nucleoside-5'-triphosphates (pH 7.0) of anti-M. luteus activity was defined as the (Sigma) 50 ^mol, tylosin tartrate (Sigma) 550 ^g, amount of that when applied to a crude enzyme protein 400 jug, divalent metal 12.7-mm paper disc (Schleicher and Schuell No. cations 40 ^rnol and potassium phosphate (pH 740-E) produces a zone of growth inhibition of 6.5) 5 fimol added per ml of distilled water. 828 THE JOURNAL OF ANTIBIOTICS MAY 1989

These mixtures were adjusted to pH 7.0 with phates in the nucleotidylation of pirli- KOH,and the reactions were stirred slowly for mycin,3'4) the phosphorylation of tylosin,6) and the designated periods of time at 25°C. Using in the case of the phosphorylation of lincos- these reaction conditions, tylosin is knownto be aminides.5) Data presented in Table 2 compare inactivated through conversion to tylosin-2'-0- the coenzyme specificities of all three conver- phosphate.6) Quantitation of tylosin was per- sions. Tylosin was phosphorylated effectively formed as described for pirlimycin. in the presence of ATPor UTP, and to a lesser Divalent Metal Cation Specificity extent in the presence of GTP, while pirlimycin Previous publications from our laboratories was phosphorylated and nucleotidylated in have reported a requirement for Mg2+ in the the presence of ATP, UTP, GTP, CTP or ITP. inactivation of Iincosaminide3~5) and macrolide6) These coenzymespecificities indicate differences antibiotics as catalyzed by crude enzymeprepara- in the lincosaminide and macrolide conversion tions of S. coelicolor Miiller. As part of the enzymes. current study we have investigated the speci- Crude Enzyme Specific Activity vs. Microbial ficities of divalent metal cations in these conver- Ageat Harvest sions ofpirlimycin and tylosin. As seen in Table The specific activities related to the three con- 1, tylosin was phosphorylated in the presence versions catalyzed by the S. coelicolor Miiller of Mg2+, Ca2+, Co2+, Zn2+ or Mn2+. Pirli- mycin was phosphorylated in the presence of crude enzyme were investigated as a function of Mg2+, Ca2+ or Co2+, and nucleotidylated the age of the mycelia at harvest. Data pre- sented in Table 3 indicate 24 hours to be the in the presence of Mg2+, Ca2+, Co2+, Zn2+ or Mn2+. These differences in cation specificities optimal age of S. coelicolor Miiller for use in suggest differences in the enzymes catalyzing crude enzymepreparation. The lack of signi- lincosaminide phosphorylation and nucleoti- ficant change in crude enzymespecific activity dylation. between 24 and 72 hours in the case of the phosphorylation of tylosin would also indicate CoenzymeSpecificity differences in the enzymes catalyzing pirlimycin Previous publications have also shown re- and tylosin conversions. Note the significant quirements for various nucleoside-S'-triphos- change in specific activity during the same time

Table 1. Divalent metal cation specificity for antibiotic inactivation. Antibiotic remaining (nmol/ml) Antibiotic Reaction Metal cation 0.5hour 4hours llhours 23hours 49hours Tylosin Phosphorylation Mg2+ 587 470 335 205 190 Ca2+ 587 470 335 205 190 Co2+ 587 470 469 294 235 Zn2+ 587 470 469 294 235 Mn2+ 587 470 335 294 190 None 587 587 568 469 411 Pirlimycin Phosphorylation Mg2 + 394 49 0 0 Ca2 + 394 101 0 0 Co2+ 398 233 119 16 Zn2 + 398 374 280 350 Mn2 + 398 374 280 350 None 394 398 374 403 Pirlimycin Nucl eotidylation Mg2+ 362 0 0 Ca2 + 362 262 72 Co2 + 362 31 13 Zn2+ 362 16 ll Mn2+ 362 83 0 None 362 329 277 VOL. XLH NO. 5 THE JOURNAL OF ANTIBIOTICS 829

Table 2. Coenzymespecificity for antibiotic inactivation. Antibiotic remaining (nmol/ml) Antibio tic Reaction Co enzyme 0.5hour 4hours llhours 23hours 49hours 6 ATP 585 548 205 14 41 Tylosin Phosphorylati on 0 UTP 585 548 293 19 59 6 GTP 585 548 410 29 235 0 CTP 577 552 431 41 330 0 ITP 577 552 431 41 330 9 None 577 552 504 46 469 0 ATP 559 280 56 Pirlimycin Phosphorylati o n 5 UTP 559 394 197 0 GTP 559 454 183 0 CTP 559 454 56 0 ITP 559 454 47 0 None 559 559 559 Pirlimyci n Nucleotidylation ATP 559 0 0 0 - UTP 559 215 0 0 - GTP 559 27 0 0 - CTP 559 141 0 0 - ITP 559 0 0 0 - None 559 559 559 559 -

Table 3. Crude enzyme specific activity vs. microbial age at harvest. Harvest(hours) SubstrateQn hcwe RReactionea ction (nmol/hour/mgSpecific activityprotein) 24 Pirlimycin Nucleo tidylation 233 Pirlimycin Pho sphorylation 66 Tylosin Physphorylation 37 Pirlimycin Nucleotidyl ation 18 Pirlimycin Phosphorylation 20 Tylosin Pho sphorylation 25 Pirlimycin Nucleotidylatio n 3 Pirl imycin Pho sphorylation 32 Tylosin Pho sphorylation 36 period concerning the nucleotidylation of 1: 17-21, 1986 pirlimycin and the smaller, but significant change Patt, T.E.; A.D. Argoudelis & V.P. concerning the phosphorylation of pirlimycin Marshall (Upjohn): Process for preparing lincomycin and clindamycin ribonucleotides. between 24 and 48 hours (Table 3). U.S. 4,430,495, Feb. 7, 1984 Coats, J. H. & A. D. Argoudelis: Microbial References transformation of antibiotics : Phosphorylation 1) Argoudelis, A.D. & J. H. Coats: Microbial of clindamycin by Streptomyces coelicolor, transformation of antibiotics. V. Clindamycin Mullen J. Bacteriol. 108: 459-464, 1971 ribonucleotides. J. Am. Chem. Soc. 93: 534~ Wiley, P. F.; L. Baczynskyj, L. A. Dolak, 535, 1971 J. I. Cialdella & V. P. Marshall: Enzymatic 2) Argoudelis, A.D.; J.H. Coats & S.A. phosphorylation of macrolide antibiotics. J. Mizsak: Microbial transformations of anti- Antibiotics 40: 195-201, 1987 biotics. Clindamycin ribonucleotides. J. Anti- Marshall, V.P.; J.I. Cialdella, L. biotics 30: 474-487, 1977 Baczynskyj, W. F. Liggett & R. A. Johnson: 3) Marshall, V.P.; T.E. Patt & A.D. Microbial 0-phosphorylation of macrolide anti- Argoudelis: Enzymic nucleotidylation of biotics. J. Antibiotics 42: 132-134, 1989 lincosaminide antibiotics. J. Ind. Microbiol. Hey, A. E. & A.D. Elbein: Partial purifica- 830 THE JOURNAL OF ANTIBIOTICS MAY 1989

tion and properties of a trehalase from Strepto- dye binding. Anal. Biochem. 72: 248~254, myces hygroscopicus. J. Bacteriol. 96: 105~ 1976 110, 1968 Brisson-Noel, A.; P. Delrieu, D. Samain & Bradford, M.M.: A rapid and sensitive P. Courvalin: Inactivation of lincosaminide method for quantitation of microgram quantities antibiotics in Staphylococcus. J. Biol. Chem. of protein utilizing the principle of protein- 263: 15880-15887, 1988