Microbial Resistance to Mitomycins Involves a Redox Relay Mechanism

Johnson, David A.; August, Paul R.; Shackleton, Cedric; Liu, Hung‐wen; Sherman, David H.

doi:10.1021/ja963880j

Cited by 31 publications

(27 citation statements)

References 16 publications

(12 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The presence of MC-DNA cross-links can be detected by a number of techniques, including standard agarose gel electrophoresis. We exploited the ability of the flavoreductase E 3 (31) (see Materials and Methods) to transfer electrons from NADH to MC in the design of an in vitro assay (23) to measure MRD activity. Various concentrations of MRD were incubated with constant concentrations of all other reaction components (MC, NADH, E 3 , and DNA).…”

Section: Vol 179 1997 Mitomycin-binding Drug Resistance Mechanismmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

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

et al. 1997

View full text Add to dashboard Cite

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...

show abstract

“…The use of multiple resistance mechanisms against a single toxic product is not uncommon (10). Streptomyces lavendulae, for example, protects itself against the deleterious effects of mitomycin via three distinct modes: drug inactivation by a flarin adenine dinucleotide-dependent oxidoreductase (34), drug export (51), and drug sequestration (52). That S. coelicolor employs multiple mechanisms to prevent self-destruction by Act would account for the fact that the actII-ORF2/ORF3 actVA-ORF1 triple mutant (which is defective in exporting Act) is viable (5), as is the soxR mutant.…”

Section: Discussionmentioning

confidence: 99%

Expression of the Streptomyces coelicolor SoxR Regulon Is Intimately Linked with Actinorhodin Production

et al. 2010

View full text Add to dashboard Cite

The [2Fe-2S]-containing transcription factor SoxR is conserved in diverse bacteria. SoxR is traditionally known as the regulator of a global oxidative stress response in Escherichia coli, but recent studies suggest that this function may be restricted to enteric bacteria. In the vast majority of nonenterics, SoxR is predicted to mediate a response to endogenously produced redox-active metabolites. We have examined the regulation and function of the SoxR regulon in the model antibiotic-producing filamentous bacterium Streptomyces coelicolor. Unlike the E. coli soxR deletion mutant, the S. coelicolor equivalent is not hypersensitive to oxidants, indicating that SoxR does not potentiate antioxidant defense in the latter. SoxR regulates five genes in S. coelicolor, including those encoding a putative ABC transporter, two oxidoreductases, a monooxygenase, and a possible NAD-dependent epimerase/dehydratase. Expression of these genes depends on the production of the benzochromanequinone antibiotic actinorhodin and requires intact [2Fe-2S] clusters in SoxR. These data indicate that actinorhodin, or a redox-active precursor, modulates SoxR activity in S. coelicolor to stimulate the production of a membrane transporter and proteins with homology to actinorhodin-tailoring enzymes. While the role of SoxR in S. coelicolor remains under investigation, these studies support the notion that SoxR has been adapted to perform distinct physiological functions to serve the needs of organisms that occupy different ecological niches and face different environmental challenges.

show abstract

“…The antitumor antibiotic mitomycin C (MC) produced by S. lavendulae is a bioreductively activated alkylating agent that crosslinks DNA at 5'CpG sequences. It has been widely used clinically for antitumor therapy (Johnson et al, 1997). A high molecular weight transglutaminase inhibitor has also been purified from the culture filtrate of S. lavendulae Y-200 (Ikura et al, 2000).…”

Section: Insecticidal and Larvicidal Properties Of Actinomycetesmentioning

confidence: 99%

Microbial agents against Helicoverpa armigera: Where are we and where do we need to go?

Vijayabharathi¹,

Kumari²,

Gopalakrishnan³

2014

Afr. J. Biotechnol.

View full text Add to dashboard Cite

Plants are prone to various biotic stresses in nature by bacteria, viruses, fungi, parasites, harmful insects and weeds. The biggest percentage loss (70%) in plants is attributed to insects. Lepidoptera is one such diversified phytophagous insect group, which include Helicoverpa armigera, a key pest of many food crops including chickpea, pigeonpea, pea, lentil, chillies, sunflower, tomato, tobacco and cotton. Controlling this insect has been a big task for farmers leading to the manufacture of a plethora of pesticides. However, over reliance on chemical pesticides has resulted in problems including safety risks, environmental contamination, outbreaks of secondary pests, insecticide resistance and decrease in biodiversity. Hence, there is an urgent need for the development of eco-friendly methods such as entomopathogens, antagonist or competitor populations of a third organism and botanicals to suppress H. armigera. Also, many compounds from microorganisms have been found to be effective in crop production, and these have a role in controlling H. armigera. The actinomycetes play an astounding role in controlling the key plant pathogens. They are the representative genera of higher microbial mass in the soil. Numerous studies have shown that these productive actino-bacteria can generate an impressive array of secondary metabolites such as antibiotics, antitumor agents, insecticides etc. This review emphasizes the mechanism behind resistance to insecticides along with actinomycetes and its potential as a biocontrol agent against H. armigera.

show abstract

“…Previously, three genetic loci have been identified to be involved in the cellular self-protection against MC in this microorganism. One locus (mcrA), which is located outside of the MC biosynthetic gene cluster, encodes a protein (MCRA) that reoxidizes the reductively activated MC species to the prodrug through a redox relay mechanism (5,6). Two other loci (mrd and mct) were found within the MC biosynthetic gene cluster and encode a small soluble protein (MRD) and a membrane-associated protein (MCT), respectively (7,8).…”

Section: Self-protection In the Mitomycin C (Mc)-producing Microorganismmentioning

confidence: 99%

Characterization of a quinone reductase activity for the mitomycin C binding protein (MRD): Functional switching from a drug-activating enzyme to a drug-binding protein

He¹

2001

Proceedings of the National Academy of Sciences

View full text Add to dashboard Cite

Self-protection in the mitomycin C (MC)-producing microorganismStreptomyces lavendulae includes MRD, a protein that binds MC in the presence of NADH and functions as a component of a unique drug binding-export system. Characterization of MRD revealed that it reductively transforms MC into 1,2-cis-1-hydroxy-2,7-diaminomitosene, a compound that is produced in the reductive MC activation cascade. However, the reductive reaction catalyzed by native MRD is slow, and both MC and the reduced product are bound to MRD for a relatively prolonged period. Gene shuffling experiments generated a mutant protein (MRDE55G) that conferred a 2-fold increase in MC resistance when expressed in Escherichia coli. Purified MRDE55G reduces MC twice as fast as native MRD, generating three compounds that are identical to those produced in the reductive activation of MC. Detailed amino acid sequence analysis revealed that the region around E55 in MRD strongly resembles the second active site of prokaryotic catalase-peroxidases. However, native MRD has an aspartic acid (D52) and a glutamic acid (E55) residue at the positions corresponding to the catalytic histidine and a nearby glycine residue in the catalaseperoxidases. Mutational analysis demonstrated that MRD D52H and MRDD52H/E55G conferred only marginal resistance to MC in E. coli. These findings suggest that MRD has descended from a previously unidentified quinone reductase, and mutations at the active site of MRD have greatly attenuated its catalytic activity while preserving substrate-binding capability. This presumed evolutionary process might have switched MRD from a potential drug-activating enzyme into the drug-binding component of the MC export system. P roduced by soil bacterium Streptomyces lavendulae, mitomycin C (MC) is a highly effective antitumor agent commonly used in combination chemotherapy regimens for the treatment of various carcinomas (1, 2). As a prodrug, MC does not have significant cytotoxity; however, upon enzymatic or chemical reductive activation, it will generate highly reactive DNA-alkylating agents that cause lethal intrastrand and interstrand DNA cross-links (3). The intracellular activation of MC is catalyzed by endogenous flavoreductases and proceeds by either anaerobic one electron reduction or oxygen-independent two-electron reduction (Fig. 1). A key step in both routes is the enzymatic reduction of the quinone moiety of MC, which initiates a cascade of spontaneous transformations that result in the production of a reactive quinone methide intermediate (4) and a group of reduced MC metabolites (5, 6, and 7) called mitosenes (1, 2).Activated MC maintains a strong propensity to interact covalently with DNA at 5Ј-CpG-3Ј sequences. Despite the high percentage of G ϩ C in its chromosome, S. lavendulae exhibits extraordinary resistance against MC (4). Previously, three genetic loci have been identified to be involved in the cellular self-protection against MC in this microorganism. One locus (mcrA), which is located outside of the MC biosynthetic gene cluste...

show abstract

Microbial Resistance to Mitomycins Involves a Redox Relay Mechanism

Cited by 31 publications

References 16 publications

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

Expression of the Streptomyces coelicolor SoxR Regulon Is Intimately Linked with Actinorhodin Production

Microbial agents against Helicoverpa armigera: Where are we and where do we need to go?

Characterization of a quinone reductase activity for the mitomycin C binding protein (MRD): Functional switching from a drug-activating enzyme to a drug-binding protein

Contact Info

Product

Resources

About