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BlmB and TlmB provide resistance to the bleomycin family of antitumor antibiotics by N-acetylating metal-free bleomycin, tallysomycin, phleomycin, and zorbamycin.

Coughlin JM, Rudolf JD, Wendt-Pienkowski E, Wang L, Unsin C, Galm U, Yang D, Tao M, Shen B - Biochemistry (2014)

Bottom Line: Intriguingly, the zbm gene cluster from Streptomyces flavoviridis ATCC21892 does not contain an N-acetyltransferase, yet ZBM is readily acetylated by BlmB and TlmB.We subsequently established that S. flavoviridis lacks the homologue of BlmB and TlmB, and ZbmA, the ZBM-binding protein, alone is sufficient to provide ZBM resistance.We further confirmed that BlmB can indeed confer resistance to ZBM in vivo in S. flavoviridis, introduction of which into wild-type S. flavoviridis further increases the level of resistance.

View Article: PubMed Central - PubMed

Affiliation: Division of Pharmaceutical Sciences, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.

ABSTRACT
The bleomycin (BLM) family of glycopeptide-derived antitumor antibiotics consists of BLMs, tallysomycins (TLMs), phleomycins (PLMs), and zorbamycin (ZBM). The self-resistant elements BlmB and TlmB, discovered from the BLM- and TLM-producing organisms Streptomyces verticillus ATCC15003 and Streptoalloteichus hindustanus E465-94 ATCC31158, respectively, are N-acetyltransferases that provide resistance to the producers by disrupting the metal-binding domain of the antibiotics required for activity. Although each member of the BLM family of antibiotics possesses a conserved metal-binding domain, the structural differences between each member, namely, the bithiazole moiety and C-terminal amine of BLMs, have been suggested to instill substrate specificity within BlmB. Here we report that BlmB and TlmB readily accept and acetylate BLMs, TLMs, PLMs, and ZBM in vitro but only in the metal-free forms. Kinetic analysis of BlmB and TlmB reveals there is no strong preference or rate enhancement for specific substrates, indicating that the structural differences between each member of the BLM family play a negligible role in substrate recognition, binding, or catalysis. Intriguingly, the zbm gene cluster from Streptomyces flavoviridis ATCC21892 does not contain an N-acetyltransferase, yet ZBM is readily acetylated by BlmB and TlmB. We subsequently established that S. flavoviridis lacks the homologue of BlmB and TlmB, and ZbmA, the ZBM-binding protein, alone is sufficient to provide ZBM resistance. We further confirmed that BlmB can indeed confer resistance to ZBM in vivo in S. flavoviridis, introduction of which into wild-type S. flavoviridis further increases the level of resistance.

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BlmB andTlmB acetylate four members of the BLM family of antitumorantibiotics, BLM B2, TLM A, PLM D1, and ZBM. HPLC analysis of in vitro enzyme reactions: (A) BLM B2 with BlmB, (B) TLMA with BlmB, (C) PLM D1 with BlmB, (D) ZBM with BlmB, (E) BLM B2 withTlmB, (F) TLM A with TlmB, (G) PLM D1 with TlmB, and (H) ZBM withTlmB. Panels A and E (BLM B2), B and F (TLM A), C and G (PLM D1),and D and H (ZBM) each show 30 min (I) and 15 min (II), 15 min (I)and 5 min (II), 60 min (I) and 30 min (II), and 10 min (I) and 5 min(II) reactions, respectively, and enzyme-boiled negative controls(III). Highlighted compounds are the antibiotic substrate (●)and acetylated product (*).
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fig2: BlmB andTlmB acetylate four members of the BLM family of antitumorantibiotics, BLM B2, TLM A, PLM D1, and ZBM. HPLC analysis of in vitro enzyme reactions: (A) BLM B2 with BlmB, (B) TLMA with BlmB, (C) PLM D1 with BlmB, (D) ZBM with BlmB, (E) BLM B2 withTlmB, (F) TLM A with TlmB, (G) PLM D1 with TlmB, and (H) ZBM withTlmB. Panels A and E (BLM B2), B and F (TLM A), C and G (PLM D1),and D and H (ZBM) each show 30 min (I) and 15 min (II), 15 min (I)and 5 min (II), 60 min (I) and 30 min (II), and 10 min (I) and 5 min(II) reactions, respectively, and enzyme-boiled negative controls(III). Highlighted compounds are the antibiotic substrate (●)and acetylated product (*).

Mentions: Although previous studies reported BlmB did not acetylate bleomycinicacid or PLM,21,32 our previous work on isolatingTLMs30 and ZBM16,31 afforded us the opportunity to examine the substrate flexibilityof BlmB comparatively with each of the four members of the BLM family.Both BlmB and TlmB were assayed using Cu(II)-BLM B2, TLM A, Cu(II)-PLMD1, or Cu(II)-ZBM as a substrate with EDTA included in each reactionmixture. After 30 min, the reactions were quenched by boiling, andCuSO4 was added to simplify HPLC analysis as the copper-boundand metal-free antibiotics elute at different retention times. HPLCanalysis of each reaction mixture confirmed that both BlmB and TlmBare capable of acetylating BLM B2, TLM A, PLM D1, and ZBM (Figure 2). Each acetylated product was confirmed by HR-MALDI-MS.


BlmB and TlmB provide resistance to the bleomycin family of antitumor antibiotics by N-acetylating metal-free bleomycin, tallysomycin, phleomycin, and zorbamycin.

Coughlin JM, Rudolf JD, Wendt-Pienkowski E, Wang L, Unsin C, Galm U, Yang D, Tao M, Shen B - Biochemistry (2014)

BlmB andTlmB acetylate four members of the BLM family of antitumorantibiotics, BLM B2, TLM A, PLM D1, and ZBM. HPLC analysis of in vitro enzyme reactions: (A) BLM B2 with BlmB, (B) TLMA with BlmB, (C) PLM D1 with BlmB, (D) ZBM with BlmB, (E) BLM B2 withTlmB, (F) TLM A with TlmB, (G) PLM D1 with TlmB, and (H) ZBM withTlmB. Panels A and E (BLM B2), B and F (TLM A), C and G (PLM D1),and D and H (ZBM) each show 30 min (I) and 15 min (II), 15 min (I)and 5 min (II), 60 min (I) and 30 min (II), and 10 min (I) and 5 min(II) reactions, respectively, and enzyme-boiled negative controls(III). Highlighted compounds are the antibiotic substrate (●)and acetylated product (*).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230324&req=5

fig2: BlmB andTlmB acetylate four members of the BLM family of antitumorantibiotics, BLM B2, TLM A, PLM D1, and ZBM. HPLC analysis of in vitro enzyme reactions: (A) BLM B2 with BlmB, (B) TLMA with BlmB, (C) PLM D1 with BlmB, (D) ZBM with BlmB, (E) BLM B2 withTlmB, (F) TLM A with TlmB, (G) PLM D1 with TlmB, and (H) ZBM withTlmB. Panels A and E (BLM B2), B and F (TLM A), C and G (PLM D1),and D and H (ZBM) each show 30 min (I) and 15 min (II), 15 min (I)and 5 min (II), 60 min (I) and 30 min (II), and 10 min (I) and 5 min(II) reactions, respectively, and enzyme-boiled negative controls(III). Highlighted compounds are the antibiotic substrate (●)and acetylated product (*).
Mentions: Although previous studies reported BlmB did not acetylate bleomycinicacid or PLM,21,32 our previous work on isolatingTLMs30 and ZBM16,31 afforded us the opportunity to examine the substrate flexibilityof BlmB comparatively with each of the four members of the BLM family.Both BlmB and TlmB were assayed using Cu(II)-BLM B2, TLM A, Cu(II)-PLMD1, or Cu(II)-ZBM as a substrate with EDTA included in each reactionmixture. After 30 min, the reactions were quenched by boiling, andCuSO4 was added to simplify HPLC analysis as the copper-boundand metal-free antibiotics elute at different retention times. HPLCanalysis of each reaction mixture confirmed that both BlmB and TlmBare capable of acetylating BLM B2, TLM A, PLM D1, and ZBM (Figure 2). Each acetylated product was confirmed by HR-MALDI-MS.

Bottom Line: Intriguingly, the zbm gene cluster from Streptomyces flavoviridis ATCC21892 does not contain an N-acetyltransferase, yet ZBM is readily acetylated by BlmB and TlmB.We subsequently established that S. flavoviridis lacks the homologue of BlmB and TlmB, and ZbmA, the ZBM-binding protein, alone is sufficient to provide ZBM resistance.We further confirmed that BlmB can indeed confer resistance to ZBM in vivo in S. flavoviridis, introduction of which into wild-type S. flavoviridis further increases the level of resistance.

View Article: PubMed Central - PubMed

Affiliation: Division of Pharmaceutical Sciences, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.

ABSTRACT
The bleomycin (BLM) family of glycopeptide-derived antitumor antibiotics consists of BLMs, tallysomycins (TLMs), phleomycins (PLMs), and zorbamycin (ZBM). The self-resistant elements BlmB and TlmB, discovered from the BLM- and TLM-producing organisms Streptomyces verticillus ATCC15003 and Streptoalloteichus hindustanus E465-94 ATCC31158, respectively, are N-acetyltransferases that provide resistance to the producers by disrupting the metal-binding domain of the antibiotics required for activity. Although each member of the BLM family of antibiotics possesses a conserved metal-binding domain, the structural differences between each member, namely, the bithiazole moiety and C-terminal amine of BLMs, have been suggested to instill substrate specificity within BlmB. Here we report that BlmB and TlmB readily accept and acetylate BLMs, TLMs, PLMs, and ZBM in vitro but only in the metal-free forms. Kinetic analysis of BlmB and TlmB reveals there is no strong preference or rate enhancement for specific substrates, indicating that the structural differences between each member of the BLM family play a negligible role in substrate recognition, binding, or catalysis. Intriguingly, the zbm gene cluster from Streptomyces flavoviridis ATCC21892 does not contain an N-acetyltransferase, yet ZBM is readily acetylated by BlmB and TlmB. We subsequently established that S. flavoviridis lacks the homologue of BlmB and TlmB, and ZbmA, the ZBM-binding protein, alone is sufficient to provide ZBM resistance. We further confirmed that BlmB can indeed confer resistance to ZBM in vivo in S. flavoviridis, introduction of which into wild-type S. flavoviridis further increases the level of resistance.

Show MeSH
Related in: MedlinePlus