Enzymatic degradation of sulphonated azo dye using purified azoreductase from facultative Klebsiella pneumoniae.

Heterologously expressed and purified azoreductase enzyme from facultative Klebsiella pneumoniae was used to degrade sulphonated azo dye. Methyl orange (MO) was used as the model dye to study the azo dye decolorization potential of the purified enzyme at different conditions. The enzyme had maximum activity at 40 °C and pH 8.0. The enzyme was observed to be thermo-stable as some enzyme activity was retained even at 80 °C. The apparent kinetic parameters, i.e., appKm and appVmax, for azoreductase using MO as a substrate were found to be 17.18 µM and 0.08/min, respectively. The purified enzyme was able to decolorize approximately 83% of MO (20 µM) within 10 min in the presence of NADH. Thus, efficient decolorization of MO was observed by the purified enzyme. The recombinant enzyme was purified approximately 18-fold with 46% yield at the end of four steps of the purification process. Enzyme was present in a tetrameric structure as confirmed by the volume at which protein was eluted in gel filtration chromatography, and the monomeric molecular mass of enzyme was found to be 23 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The dye degradation efficiency of azoreductase cloned from Klebsiella pneumoniae and purified from recombinant Escherichia coli was observed to be much higher as compared with the efficiencies of the reported azoreductases from other bacterial strains. In the present study, we report the purification and characterization of the azoreductase cloned from Klebsiella pneumoniae and expressed in Escherichia coli.

A novel cold-adapted nitroreductase from Psychrobactersp. ANT206: Heterologous expression, characterization and nitrobenzene reduction capacity.

Widely used in multiple industrial processes, nitro-aromatic compounds, especially nitrobenzene, in low temperature environment are considered as heavy pollutants. Among the disposal methods, the bioreduction method has attracted much attention. In this study, a novel cold-adapted nitroreductase gene (psntr) was cloned from Antarctic sea-ice bacteria Psychrobacter sp. ANT206. The psntr gene was 813 bp in length and encoded a protein with flavin mononucleotide (FMN) binding sites. Homology modeling was performed to obtain structural information such as the longer loops and reduced amount of hydrogen bonds, which might be related to the high catalytic efficiency of PsNTR at low temperature. The psntr gene was successfully cloned in cold-shock pCold I vector and transformed to the expression host Escherichia coli (E. coli) BL21 with the induction by isopropyl ß-D-thiogalactoside (IPTG) at low temperature (16 °C) for 24 h. The recombinant PsNTR (rPsNTR) was purified using Ni-NTA with the specific activity of 51.59 µmol/min/mg. Interestingly, rPsNTR displayed the highest activity at 25 °C and still maintained 46.9% of the activity at 0 °C. rPsNTR also exhibited the highest activity (136.4%) at 1.0 M NaCl with incredible salt tolerance. The kinetic parameters and substrates specificity analysis demonstrated that rPsNTR could reduce various nitro-aromatic compounds. Moreover, the result of the reduction capability revealed that the recombinant E. coli exhibited a maximum nitrobenzene reduction rate of 3.03 mM/h at 16 °C. These findings revealed that the characteristics of rPsNTR might make it an excellent candidate for the bioreduction of various nitro-aromatic compounds in the low temperature and high-salt wastewater.

A cofactor consumption screen identifies promising NfsB family nitroreductases for dinitrotoluene remediation.

OBJECTIVES: To survey a library of over-expressed nitroreductases to identify those most active with 2,4- and 2,6-dinitrotoluene substrates, as promising candidates for phytoremediation of soils and groundwater contaminated with poly-nitro toluene pollutants. RESULTS: To indirectly monitor dinitrotoluene reduction we implemented a nitroblue tetrazolium dye screen to compare relative rates of NADPH consumption for 58 nitroreductase candidates, over-expressed in a nitroreductase-deleted strain of Escherichia coli. Although the screen only provides activity data at a single substrate concentration, by altering the substrate concentration and duration of incubation we showed we could first distinguish between more-active and less-active enzymes and then discriminate between the relative rates of reduction exhibited by the most active nitroreductases in the collection. We observed that members of the NfsA and NfsB nitroreductase families were the most active with 2,4-dinitrotoluene, but that only members of the NfsB family reduced 2,6-dinitrotoluene effectively. Two NfsB family members, YfkO from Bacillus subtilis and NfsB from Vibrio vulnificus, appeared especially effective with these substrates. Purification of both enzymes as His6-tagged recombinant proteins enabled in vitro determination of Michaelis-Menten kinetic parameters with each dinitrotoluene substrate. CONCLUSIONS: Vibrio vulnificus NfsB is a particularly promising candidate for bioremediation applications, being ca. fivefold more catalytically efficient with 2,4-dinitrotoluene and over 26-fold more active with 2,6-dinitrotoluene than the benchmark E. coli nitroreductases NfsA and NfsB.

Identification of Enterococcus faecalis enzymes with azoreductases and/or nitroreductase activity.

BACKGROUND: Nitroreductases, NAD(P)H dependent flavoenzymes, are found in most of bacterial species. Even if Enterococcus faecalis strains seems to present such activity because of their sensitivity to nitrofurans, no enzyme has been described. Nitroreductases were separated of others reductases due to their capacity to reduce nitro compounds. They are further classified based on their preference in cofactor: NADH and/or NADPH. However, recently, azoreductases have been studied for their strong activity on nitro compounds, especially nitro pro-drugs. This result suggests a crossing in azo and nitro reductase activities. For the moment, no nitroreductase was demonstrated to possess azoreductase activity. But due to sequence divergence and activity specificity linked to substrates, activity prediction is not evident and biochemical characterisation remains necessary. Identifying enzymes active on these two classes of compounds: azo and nitro is of interest to consider a common physiological role. RESULTS: Four putative nitroreductases, EF0404, EF0648, EF0655 and EF1181 from Enterococcus faecalis V583 were overexpressed as his-tagged recombinant proteins in Escherichia coli and purified following a native or a denaturing/renaturing protocol. EF0648, EF0655 and EF1181 showed nitroreductase activity and their cofactor preferences were in agreement with their protein sequence phylogeny. EF0404 showed both nitroreductase and azoreductase activity. Interestingly, the biochemical characteristics (substrate and cofactor specificity) of EF0404 resembled the properties of the known azoreductase AzoA. But its sequence matched within nitroreductase group, the same as EF0648. CONCLUSIONS: We here demonstrate nitroreductase activity of the putative reductases identified in the Enterococcus faecalis V583 genome. We identified the first nitroreductase able to reduce directly an azo compound, while its protein sequence is close to others nitroreductases. Consequently, it highlights the difficulty in classifying these enzymes solely on the basis of protein sequence alignment and hereby the necessity to experimentally demonstrate the activity. The results provide additional data to consider a broader functionality of these reductases.

A new fluorescent probe with a large turn-on signal for imaging nitroreductase in tumor cells and tissues by two-photon microscopy.

Hypoxia is the important characteristic of solid tumors, and it may cause the bioactivity of nitroreductase (NTR) to display an elevated level. Hence, the development of effective monitoring methods of NTR in living systems is of great importance for detecting the occurrence and progress of tumors. Toward this goal, a novel two-photon fluorescence turn-on NTR probe GCTPOC-HY, based on the two-photon platform GCTPOC and the NTR recognition site p-nitrobenzyl ether, is designed and synthesized. The probe GCTPOC-HY exhibits eminent properties such as high sensitivity and selectivity, highly stable photo-stability, and low cytotoxicity. Besides, the probe responds to 1.5µg/mL NTR with a 130-fold fluorescence enhancement, which is larger than the reported two-photon fluorescent NTR probes. Moreover, the probe GCTPOC-HY is suitable for fluorescence imaging of NTR in living cells by one- and two-photon modes. Importantly, the probe GCTPOC-HY is successfully applied to monitor NTR in the tumor tissues with a significant fluorescence signal and a penetration depth of 70µm by using two-photon microscopy.

In vivo imaging and detection of nitroreductase in zebrafish by a new near-infrared fluorescence off-on probe.

A new near-infrared fluorescence off-on probe is developed and applied to fluorescence imaging of nitroreductase in zebrafish in vivo. The probe is readily prepared by connecting 4-nitrobenzene as a quenching and recognizing moiety to a stable hemicyanine skeleton that can be formed via the decomposition of IR 780. The fluorescence off-on response of the probe to nitroreductase is based on the enzyme-catalyzed reduction of the 4-nitrobenzene moiety, followed by the 1,6-rearrangement-elimination and the fluorophore release. Compared with the existing nitroreductase probes, the proposed probe exhibits superior analytical performance such as near-infrared fluorescence emission over 700 nm as well as high selectivity and sensitivity, with a detection limit of 14 ng/mL. More importantly, the probe has been successfully applied to visualize the distribution of nitroreductase in living zebrafish in vivo, revealing that nitroreductase might mainly exist in zebrafish yolk sac. The superior properties of the probe make it of great potential use in other biosystems and in vivo studies.

Spores of Clostridium engineered for clinical efficacy and safety cause regression and cure of tumors in vivo.

Spores of some species of the strictly anaerobic bacteria Clostridium naturally target and partially lyse the hypoxic cores of tumors, which tend to be refractory to conventional therapies. The anti-tumor effect can be augmented by engineering strains to convert a non-toxic prodrug into a cytotoxic drug specifically at the tumor site by expressing a prodrug-converting enzyme (PCE). Safe doses of the favored prodrug CB1954 lead to peak concentrations of 6.3 µM in patient sera, but at these concentration(s) known nitroreductase (NTR) PCEs for this prodrug show low activity. Furthermore, efficacious and safe Clostridium strains that stably express a PCE have not been reported. Here we identify a novel nitroreductase from Neisseria meningitidis, NmeNTR, which is able to activate CB1954 at clinically-achievable serum concentrations. An NmeNTR expression cassette, which does not contain an antibiotic resistance marker, was stably localized to the chromosome of Clostridium sporogenes using a new integration method, and the strain was disabled for safety and containment by making it a uracil auxotroph. The efficacy of Clostridium-Directed Enzyme Prodrug Therapy (CDEPT) using this system was demonstrated in a mouse xenograft model of human colon carcinoma. Substantial tumor suppression was achieved, and several animals were cured. These encouraging data suggest that the novel enzyme and strain engineering approach represent a promising platform for the clinical development of CDEPT.

Purification and characterization of nitroreductase from red alkaliphilic bacterium Aquiflexum sp. DL6.

Nitroaromatic compounds are toxic to living organisms. Most of them exhibit human mutagenic and carcinogenic potential. Biotransformation and bioremediation processes can convert these compounds into non-toxic compounds. Acclimatization of bacterial strain Aquiflexum sp. DL6 with nitro-aromatics resulted in significant induction of nitroreductase (EC 1.5.1.34). The enzyme was purified by the combination of DEAE-cellulose and Sephadex G-100 column chromatography with 80-fold purification and 22% yield. Molecular weight of purified nitroreductase was estimated to be 29 kDa by SDS-PAGE. The enzyme characteristics were explored by varying the pH and temperatures, and the optimum activity was found at pH 9.5 and 40 degrees C. It was revealed that the substrate specificity of nitroreductase of Aquiflexum sp. DL6 was wide for the most of the tested nitro-aromatic compounds. The kinetic parameters like Michaelis constant and velocity maxima were determined with o-nitrophenol and NADH as substrates.

Creation and screening of a multi-family bacterial oxidoreductase library to discover novel nitroreductases that efficiently activate the bioreductive prodrugs CB1954 and PR-104A.

Two potentially complementary approaches to improve the anti-cancer strategy gene-directed enzyme prodrug therapy (GDEPT) are discovery of more efficient prodrug-activating enzymes, and development of more effective prodrugs. Here we demonstrate the utility of a flexible screening system based on the Escherichia coli SOS response to evaluate novel nitroreductase enzymes and prodrugs in concert. To achieve this, a library of 47 candidate genes representing 11 different oxidoreductase families was created and screened to identify the most efficient activators of two different nitroaromatic prodrugs, CB1954 and PR-104A. The most catalytically efficient nitroreductases were found in the NfsA and NfsB enzyme families, with NfsA homologues generally more active than NfsB. Some members of the AzoR, NemA and MdaB families also exhibited low-level activity with one or both prodrugs. The results of SOS screening in our optimised E. coli reporter strain SOS-R2 were generally predictive of the ability of nitroreductase candidates to sensitise E. coli to CB1954, and of the kcat/Km for each prodrug substrate at a purified protein level. However, we also found that not all nitroreductases express stably in human (HCT-116 colon carcinoma) cells, and that activity at a purified protein level did not necessarily predict activity in stably transfected HCT-116. These results highlight a need for all enzyme-prodrug partners for GDEPT to be assessed in the specific context of the vector and cell line that they are intended to target. Nonetheless, our oxidoreductase library and optimised screens provide valuable tools to identify preferred nitroreductase-prodrug combinations to advance to preclinical evaluation.

Substrate specificity of the deazaflavin-dependent nitroreductase from Mycobacterium tuberculosis responsible for the bioreductive activation of bicyclic nitroimidazoles.

The bicyclic 4-nitroimidazoles PA-824 and OPC-67683 represent a promising novel class of therapeutics for tuberculosis and are currently in phase II clinical development. Both compounds are pro-drugs that are reductively activated by a deazaflavin (F(420)) dependent nitroreductase (Ddn). Herein we describe the biochemical properties of Ddn including the optimal enzymatic turnover conditions and substrate specificity. The preference of the enzyme for the (S) isomer of PA-824 over the (R) isomer is directed by the presence of a long hydrophobic tail. Nitroimidazo-oxazoles bearing only short alkyl substituents at the C-7 position of the oxazole were reduced by Ddn without any stereochemical preference. However, with bulkier substitutions on the tail of the oxazole, Ddn displayed stereospecificity. Ddn mediated metabolism of PA-824 results in the release of reactive nitrogen species. We have employed a direct chemiluminescence based nitric oxide (NO) detection assay to measure the kinetics of NO production by Ddn. Binding affinity of PA-824 to Ddn was monitored through intrinsic fluorescence quenching of the protein facilitating a turnover-independent assessment of affinity. Our results indicate that (R)-PA-824, despite not being turned over by Ddn, binds to the enzyme with the same affinity as the active (S) isomer. This result, in combination with docking studies in the active site, suggests that the (R) isomer probably has a different binding mode than the (S) with the C-3 of the imidazole ring orienting in a non-productive position with respect to the incoming hydride from F(420). The results presented provide insight into the biochemical mechanism of reduction and elucidate structural features important for understanding substrate binding.

Colloidal gold modified with a genetically engineered nitroreductase: toward a novel enzyme delivery system for cancer prodrug therapy.

Directed enzyme prodrug therapy is an extensive area of research in cancer chemotherapy. Although very promising, the current directed approaches are still hampered by inefficient enzyme expression and tumor targeting. This work investigates the viability of using metal nanoparticles as a novel delivery vehicle for prodrug-activating enzymes. Using genetically incorporated amino acid sequences, a nitroreductase from E. coli was directly immobilized onto a 50 nm gold colloid, as confirmed by gel electrophoresis, DLS, and UV-vis spectroscopy. The resulting conjugates showed excellent stability in changing proton and sodium chloride environments, including PBS at 37 °C. Remarkably, the immobilized nitroreductase retained more than 99% activity to the CB1954 prodrug without the need for stabilizers. This work provides the foundation for attaching prodrug-activating enzymes to metal nanoparticles for future use in directed enzyme prodrug therapy.

Structure and function of CinD (YtjD) of Lactococcus lactis, a copper-induced nitroreductase involved in defense against oxidative stress.

In Lactococcus lactis IL1403, 14 genes are under the control of the copper-inducible CopR repressor. This so-called CopR regulon encompasses the CopR regulator, two putative CPx-type copper ATPases, a copper chaperone, and 10 additional genes of unknown function. We addressed here the function of one of these genes, ytjD, which we renamed cinD (copper-induced nitroreductase). Copper, cadmium, and silver induced cinD in vivo, as shown by real-time quantitative PCR. A knockout mutant of cinD was more sensitive to oxidative stress exerted by 4-nitroquinoline-N-oxide and copper. Purified CinD is a flavoprotein and reduced 2,6-dichlorophenolindophenol and 4-nitroquinoline-N-oxide with k(cat) values of 27 and 11 s(-1), respectively, using NADH as a reductant. CinD also exhibited significant catalase activity in vitro. The X-ray structure of CinD was resolved at 1.35 A and resembles those of other nitroreductases. CinD is thus a nitroreductase which can protect L. lactis against oxidative stress that could be exerted by nitroaromatic compounds and copper.

Nitroreductase from Salmonella typhimurium: characterization and catalytic activity.

The biocatalytic activity of nitroreductase from Salmonella typhimurium (NRSal) was investigated for the reduction of alpha,beta-unsaturated carbonyl compounds, nitroalkenes, and nitroaromatics. The synthesized gene was subcloned into a pET28 overexpression system in E.coli BL21 strain, and the corresponding expressed protein was purified to homogeneity with 15% protein mass yield and 41% of total activity recovery. NRSal showed broad substrate acceptance for various nitro compounds such as 1-nitrocyclohexene and aliphatic nitroalkenes (alkene reductase activity), as well as nitrobenzene (nitroreductase activity), with substrate conversion efficiency of > 95%. However, the reduction of enones was generally low, proceeding albeit with high stereoselectivity. The efficient biocatalytic reduction of substituted nitroalkenes provides a route for the preparation of the corresponding nitroalkanes. NRSal also demonstrated the first single isolated enzyme-catalyzed reduction of nitrobenzene to aniline through the formation of nitrosobenzene and phenylhydroxylamine as intermediates. However, chemical condensation of the two intermediates to produce azoxybenzene currently limits the yield of aniline.

Correlation of the genotoxic activation and kinetic properties of Salmonella enterica serovar Typhimurium nitroreductases SnrA and cnr with the redox potentials of nitroaromatic compounds and quinones.

Bacterial nitroreductases (NRs) catalyse the oxygen-insensitive reduction of several nitro-substituted compounds and quinones. SnrA and cnr NRs have been previously identified in Salmonella enterica serovar Typhimurium; they reduce several environmental nitro compounds that display mutagenic activity in the Ames test. Although some of their biochemical properties have been reported, the substrate specificity of each protein over mutagenic nitro compounds is unknown; even more, the possible relationship between their capacity to activate nitro compounds into mutagens and the redox properties of putative substrates has been poorly investigated. We have purified SnrA and cnr and investigated their capacity to activate several mutagens in the Ames test as well as their kinetic parameters K(m) and V(max). Our results show that SnrA and cnr are able to activate 2,7-dinitrofluorene with the same efficiency and a similar mutagenic potency in the YG7132 tester strain; 1-nitropyrene and 1,3-dinitropyrene were efficiently activated by cnr, whereas 1,8-dinitropyrene, 1,6-dinitropyrene and 2-nitrofluorene were scarcely activated by either NR. The mutagenic potency of nitro compounds obtained in the presence of either enzyme correlates with their redox potential reported in the literature. On the other hand, a good correlation was obtained between the catalytic efficiency (V(max)/K(m)) of the purified cnr with the redox potential of eight molecules including nitro-substituted compounds and quinones. No correlation between redox potential and catalytic efficiency by SnrA was observed, suggesting that factors other than redox potential such as the structure of the compounds are involved in the catalytic efficiency of SnrA.

Characterization of a nitroreductase with selective nitroreduction properties in the food and intestinal lactic acid bacterium Lactobacillus plantarum WCFS1.

Nitroreductases reduce nitroaromatic compounds and other oxidants in living organisms, having interesting implications in environmental and human health. A putative nitrobenzoate reductase encoding gene (lp_0050) was recently annotated in the completed DNA sequence of lactic acid bacterium Lactobacillus plantarum WCFS1 strain. In this research, this L. plantarum gene was cloned and expressed, and the corresponding protein (PnbA) was biochemically characterized. This L. plantarum PnbA reductase is a 216 amino acid residue FMN-flavoprotein, which exhibits 23% identity with Pseudomonas putida and Ralstonia eutropha nitroreductases and <11% identity with those from enterobacteria such as E. cloacae . This reductase also showed 32-43% identity (65-72% similarity) to predicted PnbA proteins from other lactic acid bacteria. It utilized a wide range of electron acceptors including dichlorophenolindophenol (DCPIP), nitroblue tetrazolium (NBT), ferricyanide, and quinones (menadione, benzoquinone), but not pyridinium cations (paraquat and N-methyl-beta-carbolines), and it was inhibited by dicoumarol and diphenyliodonium. HPLC-MS and spectroscopic data showed that it specifically catalyzed the reduction of the 4-nitroaromatic group to the corresponding hydroxylamine in the presence of NAD(P)H. Kinetics parameters (V(max) and K(m)) showed a higher efficiency for the reduction of 2,4-dinitrobenzoate than for the reduction of 4-nitrobenzoate. It was chemoselective for the reduction of 4-nitrobenzoates, being unable to reduce other nitroaromatics. Then, L. plantarum PnbA reductase might be more specific than other microbial nitroreductases that reduce a wider range of nitroaromatic compounds. The physiological and functional role of nitroreductases remain unknown; however, their presence in lactic acid bacteria widely occurring in foods and the human intestinal tract should be of further interest.

Nitroreductase II involved in 2,4,6-trinitrotoluene degradation: purification and characterization from Klebsiella sp. Cl.

Three 2,4,6-trinitrotoluene (TNT) nitroreductases from Klebsiella sp. CI have different reduction capabilities that can degrade TNT by simultaneous utilization of two initial reduction pathways. Of these, nitroreductase II was purified to homogeneity by sequential chromatographies. Nitroreductase II is an oxygen-insensitive enzyme and reduces both TNT and nitroblue tetrazolium. The N-terminal amino acid sequence of the enzyme did not show any sequence similarity with those of other nitroreductases reported. However, it transformed TNT by the reduction of nitro groups like nitroreductase I. It had a higher substrate affinity and specific activity for TNT reduction than other nitroreductases, and it showed a higher oxidation rate of NADPH with the ortho-substituted isomers of TNT metabolites (2-hydroxylaminodinitrotoluene and 2-aminodinitrotoluene) than with para-substituted compounds (4-hydroxylaminodinitrotoluene and 4-amino-dinitrotoluene).

Expression and characterization of the TNT nitroreductase of Pseudomonas sp. HK-6 in Escherichia coli.

Pseudomonas sp. HK-6 is able to utilize 2,4,6-trinitrotoluene (TNT) as a sole nitrogen source. The pnrB gene of the HK-6 strain was cloned using degenerate primers synthesized on the basis of the sequence information of the terminal amino acids of a previously purified native TNT nitroreductase. The nucleotide sequence of pnrB was 654 bp long, and its deduced polypeptide sequence was composed of 217 amino acid residues with a predicted molecular mass of 24 kDa. To facilitate the purification and characterization of this enzyme, an Escherichia expression plasmid harboring six histidine residues fused to a pnrB gene was constructed (His6-PnrB) and designated pPSC1. The His6-PnrB induced in E. coli BL21 was purified using a nickel affinity column to homogeneity. Its enzymatic activity was assayed by measuring absorbance changes at 340 nm due to NADH oxidation. The V (max) and K ( m ) values of the enzyme for TNT were 12.6 micromol/min/mg protein and 2.9 mM, respectively. In addition, the pnrB knockout mutant was constructed via a single-crossover homologous recombination with a partial pnrB DNA fragment that lacked both start and stop codons. Eight days was required for complete degradation of 0.5 mM TNT by the wild-type HK-6 strain, whereas the pnrB mutant degraded only 10% of the TNT in the same time period. Even after 20 days, only approximately 50% of the 0.5 mM TNT was degraded by the pnrB mutant. These results illustrate that pnrB may perform a crucial role in the TNT degradation pathway of the HK-6 strain.

Crystallization and preliminary X-ray characterization of the Bacillus amyloliquefaciens YwrO enzyme.

CB1954 is an anticancer prodrug that is currently in clinical trials coupled with the Escherichia coli flavoenzyme nitroreductase (NTR) for use in directed-enzyme prodrug therapy (DEPT). The NTR enzyme is responsible for the conversion of the prodrug into a cytotoxic agent. The bifunctional alkylating agent produced by this bioactivation process leads to DNA damage and death of cancer cells. Recently, a novel flavoenzyme from Bacillus amyloliquefaciens, YwrO (Bam YwrO), was reported to be able to reduce CB1954 from its noncytotoxic form into its active form. The crystallization and preliminary X-ray diffraction analysis of two crystal forms of Bam YwrO are reported. The first crystal form is orthorhombic, with space group P22(1)2(1), and diffracts X-rays to 2.18 A resolution. The second crystal form is tetragonal, with space group P4(1), and diffracts X-rays to 3.4 A. Determination of the Bam YwrO crystal structure will provide an understanding of the molecular recognition between this enzyme and the anticancer prodrug CB1954.

TNT biotransformation: when chemistry confronts mineralization.

Our understanding of the genetics and biochemistry of microbial 2,4,6-trinitrotoluene (TNT) biotransformation has advanced significantly during the past 10 years, and biotreatment technologies have developed. In this review, we summarize this new knowledge. A number of enzyme classes involved in TNT biotransformation include the type I nitroreductases, the old yellow enzyme family, a respiration-associated nitroreductase, and possibly ring hydroxylating dioxygenases. Several strains harbor dual pathways: nitroreduction (reduction of the nitro group in TNT to a hydroxylamino and/or amino group) and denitration (reduction of the aromatic ring of TNT to Meisenheimer complexes with nitrite release). TNT can serve as a nitrogen source for some strains, and the postulated mechanism involves ammonia release from hydroxylamino intermediates. Field biotreatment technologies indicate that both stimulation of microbial nitroreduction and phytoremediation result in significant and permanent immobilization of TNT via its metabolites. While the possibility for TNT mineralization was rekindled with the discovery of TNT denitration and oxygenolytic and respiration-associated pathways, further characterization of responsible enzymes and their reaction mechanisms are required.

Repeated cycles of Clostridium-directed enzyme prodrug therapy result in sustained antitumour effects in vivo.

The unique properties of the tumour microenvironment can be exploited by using recombinant anaerobic clostridial spores as highly selective gene delivery vectors. Although several recombinant Clostridium species have been generated during the past decade, their efficacy has been limited. Our goal was to substantially improve the prospects of clostridia as a gene delivery vector. Therefore, we have assessed a series of nitroreductase (NTR) enzymes for their capacity to convert the innocuous CB1954 prodrug to its toxic derivative. Among the enzymes tested, one showed superior prodrug turnover characteristics. In addition, we established an efficient gene transfer procedure, based on conjugation, which allows for the first time genetic engineering of Clostridium strains with superior tumour colonisation properties with high success rates. This conjugation procedure was subsequently used to create a recombinant C. sporogenes overexpressing the isolated NTR enzyme. Finally, analogous to a clinical setting situation, we have tested the effect of multiple consecutive treatment cycles, with antibiotic bacterial clearance between cycles. Importantly, this regimen demonstrated that intravenously administered spores of NTR-recombinant C. sporogenes produced significant antitumour efficacy when combined with prodrug administration.