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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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(A) Conformations of free and enzyme-boundBLM reveal its structuralflexibility: (I) free Co(II)-BLM A2 (Protein Data Bank entry 1DEY), (II) Cu(II)-BLMA2 bound to BlmA (Protein Data Bank entry 1JIF), and (III) BLM A2 bound to BlmB (ProteinData Bank entry 2ZW7). Enzymes are not shown. Co(II) or Cu(II) atoms are shown as goldspheres. The five metal-coordinating nitrogens are shown as blue spheres.For II and III, the bithiazole moieties and C-terminal amines arecropped at the double wavy lines. The tight conformation of the metal-bindingdomain in metal-bound BLM likely prevents BLM from correctly bindingto BlmB for acetylation. (B) Unified model for self-resistance withinthe producers of the BLM family of antitumor antibiotics as exemplifiedby the BLM producer. Activated BLM (or TLM or ZBM) is generated bybinding to a metal ion [M(II)] and molecular oxygen (O2), which is then reduced to form BLM-M(III)-OOH (black box). Resistancein the native producer is primarily achieved through sesquestrationby BlmA (thick blue box), while antibiotic modification by BlmB (thingreen box) provides an additional mechanism of resistance. BlmB acetylatesthe axial ligand of the BLM metal-binding domain, but only in thepresence of metal-free BLM. Acetylated BLM (Ac-BLM) can bind metal,but O2 binding or activation is inhibited. BlmA tightlybinds metal-bound BLM, preventing O2 binding and activation.For the sake of simplicity, the coordinated metal ion is representedas an M. Members of the BLM family are isolated as Cu(II) complexes;structures have been determined as Cu or Co complexes, and Fe(II)must bind before activated BLM is generated.
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fig4: (A) Conformations of free and enzyme-boundBLM reveal its structuralflexibility: (I) free Co(II)-BLM A2 (Protein Data Bank entry 1DEY), (II) Cu(II)-BLMA2 bound to BlmA (Protein Data Bank entry 1JIF), and (III) BLM A2 bound to BlmB (ProteinData Bank entry 2ZW7). Enzymes are not shown. Co(II) or Cu(II) atoms are shown as goldspheres. The five metal-coordinating nitrogens are shown as blue spheres.For II and III, the bithiazole moieties and C-terminal amines arecropped at the double wavy lines. The tight conformation of the metal-bindingdomain in metal-bound BLM likely prevents BLM from correctly bindingto BlmB for acetylation. (B) Unified model for self-resistance withinthe producers of the BLM family of antitumor antibiotics as exemplifiedby the BLM producer. Activated BLM (or TLM or ZBM) is generated bybinding to a metal ion [M(II)] and molecular oxygen (O2), which is then reduced to form BLM-M(III)-OOH (black box). Resistancein the native producer is primarily achieved through sesquestrationby BlmA (thick blue box), while antibiotic modification by BlmB (thingreen box) provides an additional mechanism of resistance. BlmB acetylatesthe axial ligand of the BLM metal-binding domain, but only in thepresence of metal-free BLM. Acetylated BLM (Ac-BLM) can bind metal,but O2 binding or activation is inhibited. BlmA tightlybinds metal-bound BLM, preventing O2 binding and activation.For the sake of simplicity, the coordinated metal ion is representedas an M. Members of the BLM family are isolated as Cu(II) complexes;structures have been determined as Cu or Co complexes, and Fe(II)must bind before activated BLM is generated.

Mentions: Both BlmB and TlmB acetylate BLM B2, TLM A, PLM D1, and ZBM,butonly the metal-free forms, in vitro. Although metal-freeBLM as a requirement for BlmB activity was previously stated,22 it appeared to be anecdotal as we found no experimentalevidence in the literature. Comparisons of the BLM structures (Figure 4A),12,22,40 free and enzyme-bound, reveal a probable cause for the lack of acetylationof the metal-bound forms of BLMs by BlmB and TlmB. When metal is coordinatedto the five nitrogens of BLM, as in the case of the Cu(II)-BLM complexbound to BlmA or the Co(II)-BLM complex alone, the metal-binding domainforms a compact conformation. Conversely, BLM in BlmB must adopt anopen conformation allowing the primary amine of BLM access to acetyl-CoA.The tight conformation of BLM when bound to metal likely precludesthe correct binding orientation in BlmB necessary for catalysis andprevents BlmB from pulling apart the tightly chelated nitrogen atoms(Figure 4A).


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)

(A) Conformations of free and enzyme-boundBLM reveal its structuralflexibility: (I) free Co(II)-BLM A2 (Protein Data Bank entry 1DEY), (II) Cu(II)-BLMA2 bound to BlmA (Protein Data Bank entry 1JIF), and (III) BLM A2 bound to BlmB (ProteinData Bank entry 2ZW7). Enzymes are not shown. Co(II) or Cu(II) atoms are shown as goldspheres. The five metal-coordinating nitrogens are shown as blue spheres.For II and III, the bithiazole moieties and C-terminal amines arecropped at the double wavy lines. The tight conformation of the metal-bindingdomain in metal-bound BLM likely prevents BLM from correctly bindingto BlmB for acetylation. (B) Unified model for self-resistance withinthe producers of the BLM family of antitumor antibiotics as exemplifiedby the BLM producer. Activated BLM (or TLM or ZBM) is generated bybinding to a metal ion [M(II)] and molecular oxygen (O2), which is then reduced to form BLM-M(III)-OOH (black box). Resistancein the native producer is primarily achieved through sesquestrationby BlmA (thick blue box), while antibiotic modification by BlmB (thingreen box) provides an additional mechanism of resistance. BlmB acetylatesthe axial ligand of the BLM metal-binding domain, but only in thepresence of metal-free BLM. Acetylated BLM (Ac-BLM) can bind metal,but O2 binding or activation is inhibited. BlmA tightlybinds metal-bound BLM, preventing O2 binding and activation.For the sake of simplicity, the coordinated metal ion is representedas an M. Members of the BLM family are isolated as Cu(II) complexes;structures have been determined as Cu or Co complexes, and Fe(II)must bind before activated BLM is generated.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: (A) Conformations of free and enzyme-boundBLM reveal its structuralflexibility: (I) free Co(II)-BLM A2 (Protein Data Bank entry 1DEY), (II) Cu(II)-BLMA2 bound to BlmA (Protein Data Bank entry 1JIF), and (III) BLM A2 bound to BlmB (ProteinData Bank entry 2ZW7). Enzymes are not shown. Co(II) or Cu(II) atoms are shown as goldspheres. The five metal-coordinating nitrogens are shown as blue spheres.For II and III, the bithiazole moieties and C-terminal amines arecropped at the double wavy lines. The tight conformation of the metal-bindingdomain in metal-bound BLM likely prevents BLM from correctly bindingto BlmB for acetylation. (B) Unified model for self-resistance withinthe producers of the BLM family of antitumor antibiotics as exemplifiedby the BLM producer. Activated BLM (or TLM or ZBM) is generated bybinding to a metal ion [M(II)] and molecular oxygen (O2), which is then reduced to form BLM-M(III)-OOH (black box). Resistancein the native producer is primarily achieved through sesquestrationby BlmA (thick blue box), while antibiotic modification by BlmB (thingreen box) provides an additional mechanism of resistance. BlmB acetylatesthe axial ligand of the BLM metal-binding domain, but only in thepresence of metal-free BLM. Acetylated BLM (Ac-BLM) can bind metal,but O2 binding or activation is inhibited. BlmA tightlybinds metal-bound BLM, preventing O2 binding and activation.For the sake of simplicity, the coordinated metal ion is representedas an M. Members of the BLM family are isolated as Cu(II) complexes;structures have been determined as Cu or Co complexes, and Fe(II)must bind before activated BLM is generated.
Mentions: Both BlmB and TlmB acetylate BLM B2, TLM A, PLM D1, and ZBM,butonly the metal-free forms, in vitro. Although metal-freeBLM as a requirement for BlmB activity was previously stated,22 it appeared to be anecdotal as we found no experimentalevidence in the literature. Comparisons of the BLM structures (Figure 4A),12,22,40 free and enzyme-bound, reveal a probable cause for the lack of acetylationof the metal-bound forms of BLMs by BlmB and TlmB. When metal is coordinatedto the five nitrogens of BLM, as in the case of the Cu(II)-BLM complexbound to BlmA or the Co(II)-BLM complex alone, the metal-binding domainforms a compact conformation. Conversely, BLM in BlmB must adopt anopen conformation allowing the primary amine of BLM access to acetyl-CoA.The tight conformation of BLM when bound to metal likely precludesthe correct binding orientation in BlmB necessary for catalysis andprevents BlmB from pulling apart the tightly chelated nitrogen atoms(Figure 4A).

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