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PubMed2003

Nitroaryl phosphoramides as novel prodrugs for E. coli nitroreductase activation in enzyme prodrug therapy.

Cyclic and acyclic nitroaryl phosphoramide mustard analogues were activated by E. coli nitroreductase, an enzyme explored in GDEPT. The more active acyclic 4-nitrobenzyl phosphoramide mustard (7) showed 167 500x selective cytotoxicity toward nitroreductase-expressing V79 cells with an IC(50) as low as 0.4 nM. This is about 100x more active and 27x more selective than CB1954 (1). The superior activity was attributed to its better substrate activity (k(cat)/K(m) 19x better than 1) and/or excellent cytotoxicity of phosphoramide mustard released.

Animals↗
PubMed2004

Escherichia coli-nitroreductase suicide gene control of human telomerase reverse transcriptase-transduced minor histocompatibility antigen-specific cytotoxic T cells.

Adoptive immunotherapy with ex vivo generated cytotoxic T lymphocytes (CTLs) is applied for the treatment of leukemia relapses or viral infections after allogeneic stem cell transplantation. A common problem of adoptive immunotherapy strategies is the ex vivo expansion of the generated T cells to sufficient numbers. CTLs can be efficiently expanded by ectopic expression of the human telomerase gene (hTert). However, hTert transduction may also increase the chance for malignant transformation. Therefore, we explored the feasibility of suicide gene control of ex vivo generated CTLs expanded through the ectopic expression of hTert. To this end, we compared the efficacy of the new Escherichia coli-nitroreductase (E. coli-Ntr) suicide gene with the well-known herpes simplex virus-thymidine kinase (HSV-Tk). Introduction of hTert provided the transduced CTLs with a distinct growth advantage over the nontransduced CTLs. The hTert-E. coli-Ntr double-transduced CTLs retained their antigen-specific functions. Treatment of hTert-E. coli-Ntr double-transduced CTLs with metronidazole significantly inhibited the proliferation to a similar extent to the treatment of hTert-HSV-Tk double-transduced CTLs with ganciclovir. This is the first application of the E. coli-nitroreductase gene for the elimination of human T cells with metronidazole.

DNA-Binding Proteins↗
PubMed1999

Nitroreductase-mediated cell ablation is very rapid and mediated by a p53-independent apoptotic pathway.

Nitroreductase (NTR)-mediated selective cell ablation using the prodrug CB1954 has been achieved in vivo by targeting the nitroreductase gene to the luminal cells of the mammary gland in transgenic mice. We report that the cell ablation occurs very rapidly, starting as early as 7 h after administration of the prodrug. By cross-breeding the BLG-NTR transgenic mice to a p53-deficient mouse strain, we have generated BLG-NTR transgenic mice on a p53 null background and tested NTR-mediated cell ablation in these mice. The transgenic mice lacking a functional p53 gene showed cell ablation at a similar level compared with p53 wild-type transgenic mice, showing that functional p53 is not required for CB1954-NTR mediated cell death. These results provide further support for using this system in anti-cancer therapy.

Animals↗
PubMed2003

Selective ablation of human cancer cells by telomerase-specific adenoviral suicide gene therapy vectors expressing bacterial nitroreductase.

Reactivation of telomerase maintains telomere function and is considered critical to immortalization in most human cancer cells. Elevation of telomerase expression in cancer cells is highly specific: transcription of both RNA (hTR) and protein (hTERT) components is strongly upregulated in cancer cells relative to normal cells. Therefore, telomerase promoters may be useful in cancer gene therapy by selectively expressing suicide genes in cancer cells and not normal cells. One example of suicide gene therapy is the bacterial nitroreductase (NTR) gene, which bioactivates the prodrug CB1954 into an active cytotoxic alkylating agent. We describe construction of adenovirus vectors harbouring the bacterial NTR gene under control of the hTR or hTERT promoters. Western blot analysis of NTR expression in normal and cancer cells infected with adenoviral vectors showed cancer cell-specific nitroreductase expression. Infection with adenoviral telomerase-NTR constructs in a panel of seven cancer cell lines resulted in up to 18-fold sensitization to the prodrug CB1954, an effect that was retained in two drug-resistant ovarian lines. Importantly, no sensitization was observed with either promoter in any of the four normal cell strains. Finally, an efficacious effect was observed in cervical and ovarian xenograft models following single intratumoural injection with low doses of vector, followed by injection with CB1954.

Adenocarcinoma↗
PubMed1998

Metronidazole resistance in Helicobacter pylori is due to null mutations in a gene (rdxA) that encodes an oxygen-insensitive NADPH nitroreductase.

Metronidazole (Mtz) is a critical component of combination therapies that are used against Helicobacter pylori, the major cause of peptic ulcer disease. Many H. pylori strains are Mtz resistant (MtzR), however, and here we show that MtzR results from loss of oxygen-insensitive NADPH nitroreductase activity. The underlying gene (called 'rdxA') was identified in several steps: transformation of Mtz-susceptible (MtzS) H. pylori with cosmids from a MtzR strain, subcloning, polymerase chain reaction (PCR) and DNA sequencing. We also found that (i) E. coli (normally MtzR) was rendered MtzS by a functional H. pylori rdxA gene; (ii) introduction of rdxA on a shuttle vector plasmid into formerly MtzR H. pylori rendered it MtzS; and (iii) replacement of rdxA in MtzS H. pylori with an rdxA::camR null insertion allele resulted in a MtzR phenotype. The 630 bp rdxA genes of five pairs of H. pylori isolates from infections that were mixed (MtzR/MtzS), but uniform in overall genotype, were sequenced. In each case, the paired rdxA genes differed from one another by one to three base substitutions. Typical rdxA genes from unrelated isolates differ by 5% in DNA sequence. Therefore, the near identity of rdxA genes from paired MtzR and MtzS isolates implicates de novo mutation, rather than horizontal gene transfer in the development of MtzR. Horizontal gene transfer could readily be demonstrated under laboratory conditions with mutant rdxA alleles. RdxA is a homologue of the classical nitroreductases (CNRs) of the enteric bacteria, but differs in cysteine content (6 vs. 1 or 2 in CNRs) and isoelectric point (pI=7.99 vs. 5.4-5.6), which might account for its reduction of low redox drugs such as Mtz. We suggest that many rdxA (MtzR) mutations may have been selected by prior use of Mtz against other infections. H. pylori itself is an early risk factor for gastric cancer; the possibility that its carcinogenic effects are exacerbated by Mtz use, which is frequent in many societies, or the reduction of nitroaromatic compounds to toxic, mutagenic and carcinogenic products, may be of significant concern in public health.

Amino Acid Sequence↗
PubMed2005

Structural and mechanistic studies of Escherichia coli nitroreductase with the antibiotic nitrofurazone. Reversed binding orientations in different redox states of the enzyme.

The antibiotics nitrofurazone and nitrofurantoin are used in the treatment of genitourinary infections and as topical antibacterial agents. Their action is dependent upon activation by bacterial nitroreductase flavoproteins, including the Escherichia coli nitroreductase (NTR). Here we show that the products of reduction of these antibiotics by NTR are the hydroxylamine derivatives. We show that the reduction of nitrosoaromatics is enzyme-catalyzed, with a specificity constant approximately 10,000-fold greater than that of the starting nitro compounds. This suggests that the reduction of nitro groups proceeds through two successive, enzyme-mediated reactions and explains why the nitroso intermediates are not observed. The global reaction rate for nitrofurazone determined in this study is over 10-fold higher than that previously reported, suggesting that the enzyme is much more active toward nitroaromatics than previously estimated. Surprisingly, in the crystal structure of the oxidized NTR-nitrofurazone complex, nitrofurazone is oriented with its amide group, rather than the nitro group to be reduced, positioned over the reactive N5 of the FMN cofactor. Free acetate, which acts as a competitive inhibitor with respect to NADH, binds in a similar orientation. We infer that the orientation of bound nitrofurazone depends upon the redox state of the enzyme. We propose that the charge distribution on the FMN rings, which alters upon reduction, is an important determinant of substrate binding and reactivity in flavoproteins with broad substrate specificity.

Acetates↗
PubMed2005

Late expression of nitroreductase in an oncolytic adenovirus sensitizes colon cancer cells to the prodrug CB1954.

We have constructed an oncolytic adenovirus expressing the Escherichia coli nitroreductase gene nfsB from an internal ribosome entry site (IRES) in the adenovirus L5 major late transcript. The virus (Tcf-NTR) has Tcf transcription factor-binding sites in the E1A, E1B, and E4 promoters, which restrict viral replication to cells that have activation of the Wnt signaling pathway. This virus was compared with an E1B-55K-deleted virus expressing nitroreductase (NTR) from a cytomegalovirus (CMV) promoter in the E1B-55K region [CRAd-NTR(PS1217H6)]. Both viruses express NTR in colorectal cancer cell lines and show increased cytopathic effect in the presence of the prodrug CB1954. Unlike the Tcf-NTR virus, the CMV-NTR virus expresses NTR in human lung fibroblasts and sensitizes these normal cells to CB1954. The in vivo activity of the viruses was tested in SW620 xenografts in nude mice by intravenous injection of 1,011 particles of virus followed 1 week later by intraperitoneal injections of CB1954. The CMV-NTR virus produced minimal effects in this model. The median time to form 1,000-mm(3) tumors in mice treated with the Tcf-NTR virus plus CB1954 was increased from 14 to 26 days (p=0.003), but this was due mainly to the direct oncolytic effect of the virus. Combination therapy with 3 x 10(11) particles of Tcf-NTR virus (given intravenously) and the mammalian target of rapamycin (mTOR) inhibitor RAD001 (everolimus) (given orally) significantly improved survival (median, >50 days), and addition of CB1954 to this regimen further delayed tumor growth. These results show that the Tcf-NTR virus is more tumor selective and active than the CMV-NTR virus. At the level of transduction that can be achieved currently with oncolytic viruses given intravenously, drugs such as RAD001, which do not require activation by the virus, produce greater increases in efficacy than prodrugs such as CB1954.

Adenoviridae↗
PubMed1988

DNA binding by 1-nitropyrene and 1,6-dinitropyrene in vitro and in vivo: effects of nitroreductase induction.

1-Nitropyrene, the predominant nitropolycyclic aromatic hydrocarbon found in diesel exhaust, is a mutagen and tumorigen. 1,6-Dinitropyrene is present in diesel exhaust in much smaller quantities than 1-nitropyrene, but is much more mutagenic and carcinogenic. In an attempt to understand this difference in biological potencies, we have compared the extent of DNA binding by these two nitropyrenes in vivo. We have also determined the effect of 1-nitropyrene pretreatment upon the induction of nitroreductases and the subsequent DNA binding by both 1-nitropyrene and 1,6-dinitropyrene. Covalent DNA binding by 1-nitropyrene could not be detected in vivo; however, 1,6-dinitropyrene formed N-(deoxyguanosin-8-yl)-1-amino-6-nitropyrene as the major DNA adduct in rat liver, kidney, urinary bladder and mammary gland, with the highest levels being found in the bladder. The capability of liver microsomes to catalyze the oxidative metabolism of 1-nitropyrene was unchanged after treating rats with 8 mg/kg 1-nitropyrene. Cytochrome P-450, NADPH-cytochrome P-450 reductase and cytochrome b5 levels were also unchanged, while slight increases were detected in NADH-cytochrome b5 reductase and epoxide hydrase activities. Liver cytosolic and microsomal nitroreductase activities toward both 1-nitropyrene and 1,6-dinitropyrene were increased 2-fold, and cytosolic nitrosoreductase activity toward 1-nitrosopyrene and 1-nitro-6-nitrosopyrene was elevated by approximately 20%. DNA binding of both 1-nitropyrene and 1,6-dinitropyrene in vitro was 2-fold higher when using cytosol from 1-nitropyrene-pretreated rats. However, pretreatment of rats with 1-nitropyrene only slightly increased the amount of in vivo DNA binding by 1,6-dinitropyrene except in kidney where there was a 60% increase. These results indicate that although nitroreduction is involved in DNA adduct formation by 1,6-dinitropyrene, additional factors (e.g. O-acetylation) limit the extent of DNA binding in vivo.

Animals↗
PubMed1996

Gene cloning, purification, and characterization of NfsB, a minor oxygen-insensitive nitroreductase from Escherichia coli, similar in biochemical properties to FRase I, the major flavin reductase in Vibrio fischeri.

nfsB, encoding a minor oxygen-insensitive nitroreductase, was isolated by PCR using primers corresponding to two amino acid sequences conserved among the major flavin reductase from Vibrio fischeri and classical nitroreductases from Salmonella typhimurium and Enterobacter cloacae. The gene product, NfsB, was purified to homogeneity from extracts of Escherichia coli cells overexpressing it. NfsB was found to be situated at 13 min on the E. coli map. Biochemical analysis indicated NfsB to be a polypeptide having a calculated molecular weight of 23,904, capable of forming a homodimer and associated tightly with FMN as a prosthetic group. Although it exhibited a lower affinity to the NfsB apoenzyme than FMN, FAD could serve as an effective substitute for FMN. It was also shown that NfsB has a broad electron acceptor specificity and is associated with a low level of the NAD(P)H-flavin oxidoreductase. The NfsB catalysis obeys the ping pong Bi-Bi mechanism. The Km value for NADH varied depending on the second substrate used.

Amino Acid Sequence↗
PubMed1998

Isolation of nitrofurantoin-resistant mutants of nitroreductase-producing Clostridium sp. strains from the human intestinal tract.

Five spontaneous nitrofurantoin-resistant mutants (one each of Clostridium leptum, Clostridium paraputrificum, two other Clostridium spp. strains from the human intestinal microflora, and Clostridium perfringens ATCC 3626) were selected by growth on a nitrofurantoin-containing medium. All of the Clostridium wild-type and mutant strains produced nitroreductase, as was shown by the conversion of 4-nitrobenzoic acid to 4-aminobenzoic acid. High-performance liquid chromatography (HPLC) analysis of the mutants during incubation with 50 microg of nitrofurantoin per ml showed the gradual disappearance of the nitrofurantoin peak. The nitrofurantoin peak also disappeared when cell-free supernatants instead of cultures of each of the resistant and wild-type bacteria were used, but it persisted if the cell-free supernatants had been inactivated by heat. At least two of the mutants converted nitrofurantoin to metabolites without antibacterial activity, as was shown by a bioassay with a nitrofurantoin-susceptible Bacillus sp. strain. Nitrofurantoin at a high concentration (50 microg/ml) continued to exert some toxicity, even on the resistant strains, as was evident from the longer lag phases. This study indicates that Clostridium strains can develop resistance to nitrofurantoin while retaining the ability to produce nitroreductase; the mutants metabolized nitrofurantoin to compounds without antibacterial activity.

Anti-Infective Agents, Urinary↗
PubMed2003

Oxygen-insensitive nitroreductases NfsA and NfsB of Escherichia coli function under anaerobic conditions as lawsone-dependent Azo reductases.

Quinones can function as redox mediators in the unspecific anaerobic reduction of azo compounds by various bacterial species. These quinones are enzymatically reduced by the bacteria and the resulting hydroquinones then reduce in a purely chemical redox reaction the azo compounds outside of the cells. Recently, it has been demonstrated that the addition of lawsone (2-hydroxy-1,4-naphthoquinone) to anaerobically incubated cells of Escherichia coli resulted in a pronounced increase in the reduction rates of different sulfonated and polymeric azo compounds. In the present study it was attempted to identify the enzyme system(s) responsible for the reduction of lawsone by E. coli and thus for the lawsone-dependent anaerobic azo reductase activity. An NADH-dependent lawsone reductase activity was found in the cytosolic fraction of the cells. The enzyme was purified by column chromatography and the amino-terminal amino acid sequence of the protein was determined. The sequence obtained was identical to the sequence of an oxygen-insensitive nitroreductase (NfsB) described earlier from this organism. Subsequent biochemical tests with the purified lawsone reductase activity confirmed that the lawsone reductase activity detected was identical with NfsB. In addition it was proven that also a second oxygen-insensitive nitroreductase of E. coli (NfsA) is able to reduce lawsone and thus to function under adequate conditions as quinone-dependent azo reductase.

Amino Acid Sequence↗
PubMed2002

Evidence for lateral transfer of genes encoding ferredoxins, nitroreductases, NADH oxidase, and alcohol dehydrogenase 3 from anaerobic prokaryotes to Giardia lamblia and Entamoeba histolytica.

Giardia lamblia and Entamoeba histolytica are amitochondriate, microaerophilic protists which use fermentation enzymes like those of bacteria to survive anaerobic conditions within the intestinal lumen. Genes encoding fermentation enzymes and related electron transport peptides (e.g., ferredoxins) in giardia organisms and amebae are hypothesized to be derived from either an ancient anaerobic eukaryote (amitochondriate fossil hypothesis), a mitochondrial endosymbiont (hydrogen hypothesis), or anaerobic bacteria (lateral transfer hypothesis). The goals here were to complete the molecular characterization of giardial and amebic fermentation enzymes and to determine the origins of the genes encoding them, when possible. A putative giardia [2Fe-2S]ferredoxin which had a hypothetical organelle-targeting sequence at its N terminus showed similarity to mitochondrial ferredoxins and the hydrogenosomal ferredoxin of Trichomonas vaginalis (another luminal protist). However, phylogenetic trees were star shaped, with weak bootstrap support, so we were unable to confirm or rule out the endosymbiotic origin of the giardia [2Fe-2S]ferredoxin gene. Putative giardial and amebic 6-kDa ferredoxins, ferredoxin-nitroreductase fusion proteins, and oxygen-insensitive nitroreductases each tentatively supported the lateral transfer hypothesis. Although there were not enough sequences to perform meaningful phylogenetic analyses, the unique common occurrence of these peptides and enzymes in giardia organisms, amebae, and the few anaerobic prokaryotes suggests the possibility of lateral transfer. In contrast, there was more robust phylogenetic evidence for the lateral transfer of G. lamblia genes encoding an NADH oxidase from a gram-positive coccus and a microbial group 3 alcohol dehydrogenase from thermoanaerobic prokaryotes. In further support of lateral transfer, the G. lamblia NADH oxidase and adh3 genes appeared to have an evolutionary history distinct from those of E. histolytica.

Alcohol Dehydrogenase↗
PubMed1998

Conversion of NfsA, the major Escherichia coli nitroreductase, to a flavin reductase with an activity similar to that of Frp, a flavin reductase in Vibrio harveyi, by a single amino acid substitution.

NfsA is the major oxygen-insensitive nitroreductase of Escherichia coli, similar in amino acid sequence to Frp, a flavin reductase of Vibrio harveyi. Here, we show that a single amino acid substitution at position 99, which may destroy three hydrogen bonds in the putative active center, transforms NfsA from a nitroreductase into a flavin reductase that is as active as the authentic Frp and a tartrazine reductase that is 30-fold more active than wild-type NfsA.

Amino Acid Sequence↗
PubMed2001

Roles of FrxA and RdxA nitroreductases of Helicobacter pylori in susceptibility and resistance to metronidazole.

The relative importance of the frxA and rdxA nitroreductase genes of Helicobacter pylori in metronidazole (MTZ) susceptibility and resistance has been controversial. Jeong et al. (J. Bacteriol. 182:5082--5090, 2000) had interpreted that Mtz(s) H. pylori were of two types: type I, requiring only inactivation of rdxA to became resistant, and type II, requiring inactivation of both rdxA and frxA to become resistant; frxA inactivation by itself was not sufficient to confer resistance. In contrast, Kwon et al. (Antimicrob. Agents Chemother. 44:2133--2142, 2000) had interpreted that resistance resulted from inactivation either of frxA or rdxA. These two interpretations were tested here. Resistance was defined as efficient colony formation by single cells from diluted cultures rather than as growth responses of more dense inocula on MTZ-containing medium. Tests of three of Kwon's Mtz(s) strains showed that each was type II, requiring inactivation of both rdxA and frxA to become resistant. In additional tests, derivatives of frxA mutant strains recovered from MTZ-containing medium were found to contain new mutations in rdxA, and frxA inactivation slowed MTZ-induced killing of Mtz(s) strains. Northern blot analyses indicated that frxA mRNA, and perhaps also rdxA mRNA, were more abundant in type II than in type I strains. We conclude that development of MTZ resistance in H. pylori requires inactivation of rdxA alone or of both rdxA and frxA, depending on bacterial genotype, but rarely, if ever, inactivation of frxA alone, and that H. pylori strains differ in regulation of nitroreductase gene expression. We suggest that such regulatory differences may be significant functionally during human infection.

Blotting, Northern↗
PubMed1981

Type I nitroreductases of Escherichia coli.

Analysis of partially purified crude extract of Escherichia coli K12 by chromatography and gel electrophoresis has resulted in the separation of three distinct activities which catalyse the reduction of nitrofurazone (semicarbazone of 5-nitro-2-furaldehyde) in the presence of oxygen (type I nitroreductases). The major enzymatic activity (type IA), which was dependent solely on NADPH as a cofactor, was absent from nitrofurazone-resistant strains NFR 402 and NFR 502, but present in SIL 41, a strain which is only marginally resistant to the nitrofuran. The remaining nitroreductase activities (IB1 and IB2) utilize either NADH or NADPH as a cofactor. These activities coelute from DEAE-cellulose at pH 7.2, but may be differentiated by their behaviour on CM-cellulose at pH 5.8. The reductase activity missing in SIL 41 was observed in extracts of strain NFR 402 but not NFR 502. This enzyme (IB1) though retained by DEAE-cellulose had no affinity for CM-cellulose. The only reductase present in extracts of NFR 502 (a nitrofuran-resistant strain selected after two mutational events) was type IB2. This activity, also detectable in SIL 41 and NFR 402, has not been mapped genetically. An interesting feature of the type IB2 enzyme is its apparent inactivation by MnCl2 which has been routinely used as a partial purification step in the past.

Cell-Free System↗
PubMed1999

Application of Salmonella strains with altered nitroreductase and O-acetyltransferase activities to the evaluation of the mutagenicity of airborne particles.

The Ames test was applied to evaluation of the mutagenicity of month's samples of airborne particles from the center of Wrocław (SW Poland) collected in August and December 1997. The strains used for the study were TA 98, TA 100 and their derivatives: TA 98 NR, YG 1021, YG 1024, YG 1026, YG 1029, YG 1041, YG 1042. Both studied samples were mutagenic for almost all tested strains, with the exception of the August sample which did not influence the strain TA 100 without the metabolic activation with the S9 fraction. The December sample exhibited higher genotoxic activity than the August sample. Mutagenicity ratios of the strains with reduced nitroreductase and O-acetyltransferase activities were higher, and of the strain without the nitroreductase--lower than those of the parent strains. This indicates that nitro and amino derivatives of PAHs are responsible for the significant proportion of total mutagenicity of the studied samples of particulates. Metabolic activation with the S9 fraction caused the increase of the mutagenic activity of the samples, which indicates the presence of promutagens. The GC-MS analysis revealed the presence of known indirect mutagens from the PAHs group.

Acetyltransferases↗
PubMed1992

[The role of bacterial metabolism in transformation of non-mutagenic compounds into mutagens. I. Participation of Escherichia coli nitroreductase in creation of mutagens from non-mutagenic new pesticides].

Investigations concerned Escherichia coli nitroreductase in creation of mutagens from non-mutagenic pesticides-derivatives of urea. Three new compounds were studied: N-phenyl-N'-methylurea (IPO 4328), N-methyl,N-(2-hydroxyethyl)-N'phenylurea (IPO 2363), N-(2-hydroxyethyl), N-methyl-N'-(3,4 dichloroethyl) urea, and diurone-3-(3,4 dichlorophenyl)-1,1 dimethylurea. These compounds were incubated in anaerobic conditions with cells of E. coli K-12 (KF) strain and nitrate or nitrite. Using Ames test, mutagenicity of resulting metabolites was investigated. It was found that during incubation of herbicide IPO 4328 with cells of E. coli K-12 (KF) and nitrate, mutagenic product for strain of S. typhimurium TA 1537 is created. Very weak mutagenic metabolite for the same strain was appearing during incubation of herbicide IPO 2363 with cells of E. coli K-12 (KF) in presence of nitrite. Incubation of investigated compounds with E. coli K-12 (KF) cells alone did not result in appearance of mutagenic substances. Thus, role of Escherichia coli in creation of mutagenic compounds from non-mutagenic derivatives of urea consisted of nitrite from nitrate production with participation of nitroreductase, which afterwards in absence of bacteria or action of their enzymes reacted with investigated pesticides.

Escherichia coli↗
PubMed1991

Cloning, nucleotide sequence, and expression of the nitroreductase gene from Enterobacter cloacae.

The "classical" nitroreductases of enteric bacteria are flavoproteins which catalyze the reduction of a variety of nitroaromatic compounds to metabolites which are highly toxic, mutagenic, or carcinogenic. The gene for the nitroreductase Enterobacter cloacae has now been cloned using an antibody specific to this protein. The nucleotide sequence of the structural gene and flanking regions are reported. Sequence analysis indicates that this gene belongs to a gene family of flavoproteins which have not been previously described. Analysis of the 5'-untranslated region reveals the presence of putative regulatory elements which may be involved in the modulation of the expression of this enzyme. The cloned gene was placed under the control of a T7 promoter for overexpression of the protein in Escherichia coli. The expressed recombinant protein was purified to homogeneity and exhibited physical, spectral, and catalytic properties identical to the protein isolated from E. cloacae.

Amino Acid Sequence↗