Reductive metabolism and protein binding of chromium(VI) by P450 protien enzymes

Mikalsen, Arne; Alexander, Jan; Wallin, Håkan; Ingelman‐Sundberg, Magnus; Ra, Andersen

doi:10.1093/carcin/12.5.825

Cited by 46 publications

(24 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It was also suggested that Cr(V) is actually a toxic and mutagenic agent (2,15,16). Some reports showed that glutathione reductase (22), DT-diaphorase (7), aldehyde oxidase (3), and cytochrome P-450 (8,19,23) have Cr(VI)-reducing activity. However, it is still unclear whether these enzymes contribute to the detoxication or rather to the toxification.…”

Section: Resultsmentioning

confidence: 99%

NAD(P)H-dependent chromium (VI) reductase of Pseudomonas ambigua G-1: a Cr(V) intermediate is formed during the reduction of Cr(VI) to Cr(III)

et al. 1992

View full text Add to dashboard Cite

An NAD(P)H-dependent Cr(VI) reductase (molecular weight = 65,000) was purified from a Cr(VI)-resistant bacterium, Pseudomonas ambigua G-1. Stoichiometric analysis of the enzymatic reaction showed that the enzyme catalyzed the reduction of 1 mol of Cr(VI) to Cr(III) while consuming 3 mol of NADH as an electron donor. Chromium(VI) was reduced to Cr(V) by one equivalent NADH molecule in the absence of the enzyme.Electron spin resonance analysis showed that Cr(V) species (g = 1.979) was formed during the enzymatic reduction. The amount of Cr(V) species formed was about 10 times larger than that of the nonenzymatic reduction. These findings show that the Cr(VI) reductase reduced Cr(VI) to Cr(HI) (14) found NAD(P)H-dependent Cr(VI)-reducing activity in the cell-free supernatant fluids from Pseudomonas putida. Das and Chandra (6) have reported that Cr(VI) was reduced to Cr(III) in the presence of NAD(P)H in the cell extract of Streptomyces species. A membrane-associated chromate reduction system was also reported. Ohtake and coworkers (20) and Wang and coworkers (26)(27)(28) have shown that Cr(VI) was reduced to Cr(III) under anaerobic conditions by Enterobacter cloacae and its reduction was caused by the respiratory chain system of the cell membrane. We considered that these bacterial Cr(VI) reduction systems contribute to its detoxification by Cr(VI)-resistant bacteria. However, the Cr(VI) reductase has not yet been purified, and the details of the reaction mechanism are still unclear.P. ambigua G-1 was able to grow in medium containing up to 20 mM K2CrO4 (13). The Cr(VI)-reducing activity (11) of this strain was more thermostable than that of other pseudomonads (14). The Cr(VI)-sensitive mutants of this strain lost the reducing activity (12). These results indicate that this strain is a good source to investigate the Cr(VI) reduction mechanism which contributes to Cr(VI) detoxication.In the present paper, we report the purification of NAD(P)H-dependent Cr(VI) reductases of P. ambigua G-1 and the analysis of the catalytic mechanism of Cr(VI) reduction by the enzymes. Preparation of Cr(VI) reductase. Cells grown in 4 liters of L broth at 37°C were harvested at the late logarithmic phase, washed twice with 20 mM Tris-HCl, pH 7.2, suspended in 100 ml of the same buffer, and disrupted by sonication. After removal of the unbroken cells by centrifugation (1,500 x g, 30 min), the supernatant fluid was heat treated at 60°C for 2 min and centrifuged (1,500 x g, 30 min). The supernatant fluid was separated on a DE32 column (3.5 by 6 cm; Whatman Ltd., Maidstone, England) previously equilibrated with buffer A (50 mM glucose in 20 mM Tris-HCl, pH 7.2), and proteins were eluted with 0.4 M NaCl in buffer A at a flow rate of 100 ml/h. The fractions carrying the Cr(VI)-reducing activity were concentrated and separated by Cu2" chelate affinity chromatography using an AF-chelate Toyopearl 650M column (2.6 by 4 cm; Toso Ltd., Tokyo, Japan) which was sequentially equilibrated with 200 PM CUSO4 solution and buffer B (0.5 M NaCl in buf...

show abstract

Section: Resultsmentioning

confidence: 99%

NAD(P)H-dependent chromium (VI) reductase of Pseudomonas ambigua G-1: a Cr(V) intermediate is formed during the reduction of Cr(VI) to Cr(III)

et al. 1992

View full text Add to dashboard Cite

show abstract

“…Chromate-reducing activities have been associated with DT-diaphorase (7) and aldehyde oxidase (1) in the cell cytoplasm. Cytochrome P450 located in the cell membrane is also known to have chromate-reducing activity (21). It seems that various reductases in the cell can function in chromate reduction.…”

mentioning

confidence: 99%

“…Type I is oxygen insensitive (3,6,15,17,20,37,38,39), and type II is oxygen sensitive (21,26). In type I, there are two nitroflavin reductase superfamiles: NfsA and NfsB (3,6,14,17,20,37,38,39).…”

mentioning

confidence: 99%

Vibrio harveyi Nitroreductase Is Also a Chromate Reductase

Kwak

Lee

Kim

2003

Appl Environ Microbiol

144

View full text Add to dashboard Cite

The chromate reductase purified from Pseudomonas ambigua was found to be homologous with several nitroreductases. Escherichia coli DH5␣ and Vibrio harveyi KCTC 2720 nitroreductases were chosen for the present study, and their chromate-reducing activities were determined. A fusion between glutathione Stransferase (GST) and E. coli DH5␣ NfsA (GST-EcNfsA), a fusion between GST and E. coli DH5␣ NfsB (GST-EcNfsB), and a fusion between GST and V. harveyi KCTC 2720 NfsA (GST-VhNfsA) were prepared for their overproduction and easy purification. GST-EcNfsA, GST-EcNFsB, and GST-VhNFsA efficiently reduced nitrofurazone and 2,4,6-trinitrotoluene (TNT) as their nitro substrates. The K m values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 11.8, 23.5, and 5.4 M, respectively. The V max values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA were 3.8, 3.9, and 10.7 nmol/min/mg of protein, respectively. GST-VhNfsA was the most effective of the three chromate reductases, as determined by each V max /K m value. The optimal temperatures of GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 55, 30, and 30°C, respectively. Thus, it is confirmed that nitroreductase can also act as a chromate reductase. Nitroreductases may be used in chromate remediation. GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA have a molecular mass of 50 kDa and exist as a monomer in solution. Thin-layer chromatography showed that GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA contain FMN as a cofactor. GST-VhNfsA reduced Cr(VI) to Cr(III). Cr(III) was much less toxic to E. coli than Cr(VI).Environmental pollution by chromium may be severe. Chromium contamination is known to be prevalent at U.S. Department of Energy sites (29). The electroplating and leather-tanning industries also contribute to environmental contamination with Cr(VI) (23). Chromate compounds containing Cr(VI) are used widely in the cooling towers of heavy industry and atomic power plants, since Cr(VI) prevents corrosion and the growth of organisms (2). Cr(VI) is soluble, toxic, and carcinogenic, whereas Cr(III) is less soluble and less toxic (12). Thus, it is desirable to change Cr(VI) into Cr(III). This approach is taken in the bioremediation of Cr(VI) pollution. It shows promise for solving pollution problems and has advantages over various other physical and chemical methods.Chromate-reducing activities can be found in the cell extracts of many bacteria (4,5,8,10,13,18,25,30,(33)(34)(35). Chromate reductase can reduce the toxicity of Cr(VI) by reducing it to Cr(III) and lowering its solubility (5, 9). The chromate reductase from Pseudomonas ambigua has been purified and characterized (33). Chromate-reducing activities have been associated with DT-diaphorase (7) and aldehyde oxidase (1) in the cell cytoplasm. Cytochrome P450 located in the cell membrane is also known to have chromate-reducing activity (21). It seems that various reductases in the cell can function in chromate reduction. P. ambigua chromate reductase (33) has high homology with Escherichia coli NfsA (59%) ...

show abstract

“…4 In contrast to cigarette smoke, chromium is a unitary carcinogen, although it shows different effects according to its oxidation state. 5 Most chromate particles are 1-3 m in diameter. 6 Chromium-induced lung carcinoma is clinically similar to cigarette smoke-induced lung carcinoma in four ways.…”

mentioning

confidence: 99%

Microscopic analysis of chromium accumulation in the bronchi and lung of chromate workers

Kondo

Takahashi

Ishikawa

et al. 2003

Cancer

View full text Add to dashboard Cite

BACKGROUNDIt is known that chromium is an inhaled carcinogen and an important risk factor in the development of lung carcinoma.METHODSThe authors used a microscopic X‐ray fluorescence analyzer with transmitted X‐ray mapping imaging (Horiba, Kyoto, Japan) to measure the accumulation of chromium in 10 resected lung tissue specimens and 90 biopsy specimens from chromate workers.RESULTSThe maximum chromium accumulation (mean ± standard deviation) in 10 resected lung tissue specimens was 197 ± 238 counts per second (cps)/mili ampere (mA) (range, 4–649 cps/mA). Chromium accumulation was scattered in six tissue specimens and diffuse in one specimen. Chromium accumulation in the proximal bronchi was less than in the bronchioles or subpleural regions of the lung. Chromium accumulation was detectable in 63 (70%) of 90 biopsy specimens, and the mean accumulation was 6.5 ± 9.2 cps/mA (range, 0–46.5 cps/mA). Chromium detected in bronchial tissue specimens was deposited in the bronchial stroma but not in the epithelium. The maximum chromium accumulations in dysplasic (n = 3), squamous metaplastic (n = 10), and normal bronchial epithelia (n = 9) in chromate workers and in normal bronchial epithelia (n = 3) in non‐chromate workers were 20.2 ± 5.4, 18.3 ± 12.2, 13.2 ± 13.4, and 3.0 ± 1.8 cps/mA, respectively. The amount of chromium accumulation significantly increased according to the progression of malignant change of the bronchial epithelium (P = 0.003).CONCLUSIONSPrevious studies found that lung carcinoma with chromate exposure exhibited a variety of genetic abnormalities. Considering genetic aberrations and chromium accumulation in these premalignant lesions is useful for elucidating the process of carcinogenesis in chromium‐induced lung carcinoma. Cancer 2003. © 2003 American Cancer Society.

show abstract

Reductive metabolism and protein binding of chromium(VI) by P450 protien enzymes

Cited by 46 publications

References 0 publications

NAD(P)H-dependent chromium (VI) reductase of Pseudomonas ambigua G-1: a Cr(V) intermediate is formed during the reduction of Cr(VI) to Cr(III)

NAD(P)H-dependent chromium (VI) reductase of Pseudomonas ambigua G-1: a Cr(V) intermediate is formed during the reduction of Cr(VI) to Cr(III)

Vibrio harveyi Nitroreductase Is Also a Chromate Reductase

Microscopic analysis of chromium accumulation in the bronchi and lung of chromate workers

Contact Info

Product

Resources

About