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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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Structures of selected members of the bleomycin (BLM) family ofantitumor antibiotics. Structural differences between the antibioticsare highlighted with dotted blue boxes; the five nitrogen atoms thatcoordinate the metal ion are shown in bold and colored red, and thenitrogens acetylated by BlmB and TlmB are highlighted with a solidblue box. The four members of the BLM family selected for this studyare BLM B2, TLM A, PLM D1, and ZBM.
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fig1: Structures of selected members of the bleomycin (BLM) family ofantitumor antibiotics. Structural differences between the antibioticsare highlighted with dotted blue boxes; the five nitrogen atoms thatcoordinate the metal ion are shown in bold and colored red, and thenitrogens acetylated by BlmB and TlmB are highlighted with a solidblue box. The four members of the BLM family selected for this studyare BLM B2, TLM A, PLM D1, and ZBM.

Mentions: Bleomycins(BLMs), glycopeptide-derivedantibiotics produced by Streptomyces verticillus,are clinically relevant natural products used in chemotherapy treatmentsagainst lymphomas, squamous cell carcinomas, and testicular cancer.1−4 BLMs cause sequence-specific oxidative cleavage of double-strandedDNA through a metal-dependent mechanism.5,6 BLMs containfour functional domains (Figure 1): (i) themetal-binding domain comprised of pyrimidoblamic acid and the β-hydroxylhistidine, (ii) the DNA-binding domain consisting of the bithiazolemoiety and C-terminal amine tail in cooperation with the aminopyrimidinering, (iii) a linker region connecting the metal-binding and DNA-bindingdomains, and (iv) the disaccharide moiety responsible for cell selectivity3,7−10 and DNA cleavage activity.11 Five nitrogenatoms within the metal-binding domain (Figure 1) coordinate iron and oxygen in the active form of the antibiotic.A crystal structure of BLM bound to BlmA, the BLM-binding proteinfound in the native producer, revealed that the primary amine of theβ-aminoalanine moiety of BLM is the axial ligand to the metalion in the active BLM–metal complex form.12


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)

Structures of selected members of the bleomycin (BLM) family ofantitumor antibiotics. Structural differences between the antibioticsare highlighted with dotted blue boxes; the five nitrogen atoms thatcoordinate the metal ion are shown in bold and colored red, and thenitrogens acetylated by BlmB and TlmB are highlighted with a solidblue box. The four members of the BLM family selected for this studyare BLM B2, TLM A, PLM D1, and ZBM.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Structures of selected members of the bleomycin (BLM) family ofantitumor antibiotics. Structural differences between the antibioticsare highlighted with dotted blue boxes; the five nitrogen atoms thatcoordinate the metal ion are shown in bold and colored red, and thenitrogens acetylated by BlmB and TlmB are highlighted with a solidblue box. The four members of the BLM family selected for this studyare BLM B2, TLM A, PLM D1, and ZBM.
Mentions: Bleomycins(BLMs), glycopeptide-derivedantibiotics produced by Streptomyces verticillus,are clinically relevant natural products used in chemotherapy treatmentsagainst lymphomas, squamous cell carcinomas, and testicular cancer.1−4 BLMs cause sequence-specific oxidative cleavage of double-strandedDNA through a metal-dependent mechanism.5,6 BLMs containfour functional domains (Figure 1): (i) themetal-binding domain comprised of pyrimidoblamic acid and the β-hydroxylhistidine, (ii) the DNA-binding domain consisting of the bithiazolemoiety and C-terminal amine tail in cooperation with the aminopyrimidinering, (iii) a linker region connecting the metal-binding and DNA-bindingdomains, and (iv) the disaccharide moiety responsible for cell selectivity3,7−10 and DNA cleavage activity.11 Five nitrogenatoms within the metal-binding domain (Figure 1) coordinate iron and oxygen in the active form of the antibiotic.A crystal structure of BLM bound to BlmA, the BLM-binding proteinfound in the native producer, revealed that the primary amine of theβ-aminoalanine moiety of BLM is the axial ligand to the metalion in the active BLM–metal complex form.12

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