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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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Disk diffusion assays for Cu(II)-ZBM susceptibility ofselected S. flavoviridis recombinant strains in comparisonwith thewild-type strain. (A) SB9001 (wild-type) and SB9031 (ΔzbmA-orf38) challenged with 0, 0.1, 0.5, 1, 5, 10, and 20μg of Cu(II)-ZBM (clockwise from the top right filter, respectively).(B) SB9034 (i.e., SB9031/pBS65 expressing blmB underits putative native promoter) and SB9035 (i.e., SB9031/pBS66 expressing blmB under the ErmE* promoter) challengedwith 0, 0.1, 0.5, 1, 5, 10, and 20 μg of Cu(II)-ZBM (clockwisefrom the top right filter, respectively).
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fig3: Disk diffusion assays for Cu(II)-ZBM susceptibility ofselected S. flavoviridis recombinant strains in comparisonwith thewild-type strain. (A) SB9001 (wild-type) and SB9031 (ΔzbmA-orf38) challenged with 0, 0.1, 0.5, 1, 5, 10, and 20μg of Cu(II)-ZBM (clockwise from the top right filter, respectively).(B) SB9034 (i.e., SB9031/pBS65 expressing blmB underits putative native promoter) and SB9035 (i.e., SB9031/pBS66 expressing blmB under the ErmE* promoter) challengedwith 0, 0.1, 0.5, 1, 5, 10, and 20 μg of Cu(II)-ZBM (clockwisefrom the top right filter, respectively).

Mentions: Given the abilityof BlmB andTlmB to efficiently acetylate ZBM in vitro, it issurprising that the cloned zbm gene cluster doesnot possess a blmB/tlmB homologue.To determine if a blmB/tlmB homologueresides outside the cloned zbm gene cluster, we deletedthe zbm gene cluster from S. flavoviridis SB9001, a ZBM overproducer derived from the wild-type S.flavoviridis ATCC21892 strain,16 to afford recombinant strain S. flavoviridis SB9031;the ΔzbmA-orf38 genotype of S. flavoviridis SB9031 was confirmed by Southern analysis(Figure S7 of the Supporting Information). S. flavoviridis SB9031 is incapable of ZBM productionand eliminates the only known form of self-resistance to ZBM, theZBM-binding protein ZbmA. The susceptibility of S. flavoviridis SB9031 to Cu(II)-ZBM was determined using the disk diffusion assay.While S. flavoviridis SB9001 showed resistance upto 20 μg of ZBM per disk, the S. flavoviridis SB9031 mutant was sensitive at quantities as low as 0.5–1μg of ZBM per disk, excluding the existence of other resistanceelements on the S. flavoviridis SB9031 genome (Figure 3A).


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)

Disk diffusion assays for Cu(II)-ZBM susceptibility ofselected S. flavoviridis recombinant strains in comparisonwith thewild-type strain. (A) SB9001 (wild-type) and SB9031 (ΔzbmA-orf38) challenged with 0, 0.1, 0.5, 1, 5, 10, and 20μg of Cu(II)-ZBM (clockwise from the top right filter, respectively).(B) SB9034 (i.e., SB9031/pBS65 expressing blmB underits putative native promoter) and SB9035 (i.e., SB9031/pBS66 expressing blmB under the ErmE* promoter) challengedwith 0, 0.1, 0.5, 1, 5, 10, and 20 μg of Cu(II)-ZBM (clockwisefrom the top right filter, respectively).
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Disk diffusion assays for Cu(II)-ZBM susceptibility ofselected S. flavoviridis recombinant strains in comparisonwith thewild-type strain. (A) SB9001 (wild-type) and SB9031 (ΔzbmA-orf38) challenged with 0, 0.1, 0.5, 1, 5, 10, and 20μg of Cu(II)-ZBM (clockwise from the top right filter, respectively).(B) SB9034 (i.e., SB9031/pBS65 expressing blmB underits putative native promoter) and SB9035 (i.e., SB9031/pBS66 expressing blmB under the ErmE* promoter) challengedwith 0, 0.1, 0.5, 1, 5, 10, and 20 μg of Cu(II)-ZBM (clockwisefrom the top right filter, respectively).
Mentions: Given the abilityof BlmB andTlmB to efficiently acetylate ZBM in vitro, it issurprising that the cloned zbm gene cluster doesnot possess a blmB/tlmB homologue.To determine if a blmB/tlmB homologueresides outside the cloned zbm gene cluster, we deletedthe zbm gene cluster from S. flavoviridis SB9001, a ZBM overproducer derived from the wild-type S.flavoviridis ATCC21892 strain,16 to afford recombinant strain S. flavoviridis SB9031;the ΔzbmA-orf38 genotype of S. flavoviridis SB9031 was confirmed by Southern analysis(Figure S7 of the Supporting Information). S. flavoviridis SB9031 is incapable of ZBM productionand eliminates the only known form of self-resistance to ZBM, theZBM-binding protein ZbmA. The susceptibility of S. flavoviridis SB9031 to Cu(II)-ZBM was determined using the disk diffusion assay.While S. flavoviridis SB9001 showed resistance upto 20 μg of ZBM per disk, the S. flavoviridis SB9031 mutant was sensitive at quantities as low as 0.5–1μg of ZBM per disk, excluding the existence of other resistanceelements on the S. flavoviridis SB9031 genome (Figure 3A).

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