Inducible synthesis of the Mitomycin C resistance gene product (MCRA) from Streptomyces lavendulae

August, Paul R.; Rahn, John A.; Flickinger, Michael C.; Sherman, David H.

doi:10.1016/0378-1119(96)00172-2

Cited by 12 publications

(14 citation statements)

References 16 publications

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“…lavendulae contains at least two genetic loci for resistance to MC, mcr and mrd. Previously, cloning and analysis of mcr and its product (MCRA) were shown to involve the oxidation of activated MC (2,3,23). In contrast, MRD appears to confer resistance by binding MC as a complex that prevents drug activation.…”

Section: Discussionmentioning

confidence: 99%

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Characterization of a mitomycin-binding drug resistance mechanism from the producing organism, Streptomyces lavendulae

et al. 1997

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In an effort to characterize the diversity of mechanisms involved in cellular self-protection against the antitumor antibiotic mitomycin C (MC), DNA fragments from the producing organism (Streptomyces lavendulae) were introduced into Streptomyces lividans and transformants were selected for resistance to the drug. Subcloning of a 4.0-kb BclI fragment revealed the presence of an MC resistance determinant, mrd. Nucleotide sequence analysis identified an open reading frame consisting of 130 amino acids with a predicted molecular weight of 14,364. Transcriptional analysis revealed that mrd is expressed constitutively, with increased transcription in the presence of MC. Expression of mrd in Escherichia coli resulted in the synthesis of a soluble protein with an M r of 14,400 that conferred high-level cellular resistance to MC and a series of structurally related natural products. Purified MRD was shown to function as a drug-binding protein that provides protection against cross-linking of DNA by preventing reductive activation of MC.Streptomyces species are gram-positive soil bacteria known for their ability to produce a wide range of biologically active metabolites. In addition, many resistance genes have been cloned from these bacteria. Mechanisms of cellular self-protection include drug inactivation, target site modification, reduction of intracellular concentration via efflux, and drug binding (8). The presence of multiple modes of self-protection toward a single antibiotic is well documented (9), often with one or more resistance determinants located adjacent to the corresponding biosynthetic genes.Mitomycin C (MC) was identified in 1956 as an antibiotic produced by Streptomyces lavendulae (18) and subsequently established as an important antitumor agent (19,21). MC functions as a prodrug and requires enzymatic or chemical reduction to become a highly reactive alkylating agent (19,40). The intracellular activation of MC is specified by endogenous flavoreductases (34) and proceeds by single electron reduction to the MC semiquinone radical. The relatively long-lived semiquinone species either rearranges to an alkylating intermediate (by further reduction) or transfers an electron to molecular oxygen to generate superoxide (32). Therefore, the ability of MC to inhibit bacterial and mammalian cell growth involves the combined action of DNA alkylation and the formation of reactive oxygen species. Other naturally occurring compounds within this class include bleomycin (37), enediynes (10), and the more recently discovered dihydrobenzoxazines (25,42).Recently, a locus, mcr, that confers high-level MC resistance in S. lavendulae has been reported (2, 3). The resistance gene, mcrA, encodes a flavoenzyme (MCRA) that reoxidizes reductively activated MC (23). In another example involving a DNA damaging agent, bleomycin self-resistance has been determined by drug modification (Bat) and binding (BLMA) proteins in Streptomyces verticillus (38). Beyond these examples, little is known about bacterial resistance to the growing cl...

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Section: Discussionmentioning

confidence: 99%

“…Recently, a locus, mcr, that confers high-level MC resistance in S. lavendulae has been reported (2,3). The resistance gene, mcrA, encodes a flavoenzyme (MCRA) that reoxidizes reductively activated MC (23).…”

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Characterization of a mitomycin-binding drug resistance mechanism from the producing organism, Streptomyces lavendulae

et al. 1997

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“…T7 coliphage DNA and MCRA (essentially pure MCRA was prepared as described in detail in ref. 13) were dissolved in 20 mM Tris⅐HCl buffer containing 1 mM EDTA at pH 7.4. MC and POR stock solutions were in isopropanol at concentrations of 2.…”

Section: Methodsmentioning

confidence: 99%

“…A rabbit anti-MCRA antibody (prepared as described in ref. 13) was used at a 1:4,000 dilution to detect MCRA on nitrocellulose membranes in combination with a horseradish peroxidase-conjugated anti-rabbit antibody and the enhanced chemiluminescent visualization reagent (ECL, Amersham). Molecular weight size markers are indicated in kDa.…”

Section: Methodsmentioning

confidence: 99%

“…In contrast, a single, mitomycin-induced DNA cross-link in susceptible bacteria results in death (11). Recently, the S. lavendulae encoded mcrA gene was shown to be stimulated by MC, producing MCRA, a 54-kDa flavoprotein that confers mitomycin antibiotic resistance to recipient bacteria (12)(13)(14). The mechanism of resistance by MCRA is novel: this protein reoxidizes the reduced, cytotoxic intermediate of MC to the parent drug through a redox relay mechanism, whereby electrons are shuttled from the reduced mitomycin intermediate to the covalently bound FAD cofactor of MCRA and ultimately to molecular oxygen.…”

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Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA

Belcourt

Penketh

Hodnick

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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The mitomycin C-resistance gene, mcrA, of Streptomyces lavendulae produces MCRA, a protein that protects this microorganism from its own antibiotic, the antitumor drug mitomycin C. Expression of the bacterial mcrA gene in mammalian Chinese hamster ovary cells causes profound resistance to mitomycin C and to its structurally related analog porfiromycin under aerobic conditions but produces little change in drug sensitivity under hypoxia. The mitomycins are prodrugs that are enzymatically reduced and activated intracellularly, producing cytotoxic semiquinone anion radical and hydroquinone reduction intermediates. In vitro, MCRA protects DNA from crosslinking by the hydroquinone reduction intermediate of these mitomycins by oxidizing the hydroquinone back to the parent molecule; thus, MCRA acts as a hydroquinone oxidase. These findings suggest potential therapeutic applications for MCRA in the treatment of cancer with the mitomycins and imply that intrinsic or selected mitomycin C resistance in mammalian cells may not be due solely to decreased bioactivation, as has been hypothesized previously, but instead could involve an MCRA-like mechanism.

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Applications of Streptomyces spp. Enhanced Compost in Sustainable Agriculture

et al. 2020

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Inducible synthesis of the Mitomycin C resistance gene product (MCRA) from Streptomyces lavendulae

Cited by 12 publications

References 16 publications

Characterization of a mitomycin-binding drug resistance mechanism from the producing organism, Streptomyces lavendulae

Characterization of a mitomycin-binding drug resistance mechanism from the producing organism, Streptomyces lavendulae

Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA

Applications of Streptomyces spp. Enhanced Compost in Sustainable Agriculture

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