Dataset Information

Chromate Reductase YieF from Escherichia coli Enhances Hexavalent Chromium Resistance of Human HepG2 Cells.

ABSTRACT: Hexavalent chromium (Cr(VI)) is a serious environmental pollutant and human toxicant. Mammalian cells are very sensitive to chromate as they lack efficient chromate detoxifying strategy, e.g., chromate-reducing genes that are widely present in prokaryotes. To test whether introduction of prokaryotic chromate-reducing gene into mammalian cells could render higher chromate resistance, an Escherichia coli chromate-reducing gene yieF was transfected into human HepG2 cells. The expression of yieF was measured in stably transfected cells HepG2-YieF by quantitative RT-PCR and found up-regulated by 3.89-fold upon Cr(VI) induction. In chromate-reducing ability test, HepG2-YieF cells that harbored the reductase showed significantly higher reducing ability of Cr(VI) than HepG2 control cells. This result was further supported by the evidence of increased Cr(VI)-removing ability of crude cell extract of HepG2-YieF. Moreover, HepG2-YieF demonstrated 10% higher viability and decreased expression of GSH synthesizing enzymes under Cr(VI) stress. Subcellular localization of YieF was determined by tracing GFP-YieF fusion protein that was detected in both nucleus and cytoplasm by laser confocal microscopy. Altogether, this study successfully demonstrated that the expression of a prokaryotic Cr(VI)-reducing gene yieF endowed mammalian cell HepG2 with enhanced chromate resistance, which brought new insight of Cr(VI) detoxification in mammalian cells.

SUBMITTER: Liu X

PROVIDER: S-EPMC4490421 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Chromate Reductase YieF from Escherichia coli Enhances Hexavalent Chromium Resistance of Human HepG2 Cells.

Liu Xuan X Wu Gaofeng G Zhang Yanli Y Wu Dan D Li Xiangkai X Liu Pu P

International journal of molecular sciences 20150526 6

Hexavalent chromium (Cr(VI)) is a serious environmental pollutant and human toxicant. Mammalian cells are very sensitive to chromate as they lack efficient chromate detoxifying strategy, e.g., chromate-reducing genes that are widely present in prokaryotes. To test whether introduction of prokaryotic chromate-reducing gene into mammalian cells could render higher chromate resistance, an Escherichia coli chromate-reducing gene yieF was transfected into human HepG2 cells. The expression of yieF was ...[more]

PMID: 26016500

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Project description:Soil bacteria can detoxify Cr(VI) ions by reduction. Within the last 2 decades, numerous reports of chromate reductase enzymes have been published. These reports describe catalytic reduction of chromate ions by specific enzymes. These enzymes each have sequence similarity to known redox-active flavoproteins. We investigated the enzyme NfoR from Staphylococcus aureus, which was reported to be upregulated in chromate-rich soils and to have chromate reductase activity (H. Han, Z. Ling, T. Zhou, R. Xu, et al., Sci Rep 7:15481, 2017, https://doi.org/10.1038/s41598-017-15588-y). We show that NfoR has structural similarity to known flavin mononucleotide (FMN) reductases and reduces FMN as a substrate. NfoR binds FMN with a dissociation constant of 0.4 μM. The enzyme then binds NADPH with a dissociation constant of 140 μM and reduces the flavin at a rate of 1,350 s-1 Turnover of the enzyme is apparently limited by the rate of product release that occurs, with a net rate constant of 0.45 s-1 The rate of product release limits the rate of observed chromate reduction, so the net rate of chromate reduction by NfoR is orders of magnitude lower than when this process occurs in solution. We propose that NfoR is an FMN reductase and that the criterion required to define chromate reduction as enzymatic has not been met. That NfoR expression is increased in the presence of chromate suggests that the survival adaption was to increase the net rate of chromate reduction by facile, adventitious redox processes.IMPORTANCE Chromate is a toxic by-product of multiple industrial processes. Chromate reduction is an important biological activity that ameliorates Cr(VI) toxicity. Numerous researchers have identified chromate reductase activity by observing chromate reduction. However, all identified chromate reductase enzymes have flavin as a cofactor or use a flavin as a substrate. We show here that NfoR, an enzyme claimed to be a chromate reductase, is in fact an FMN reductase. In addition, we show that reduction of a flavin is a viable way to transfer electrons to chromate but that it is unlikely to be the native function of enzymes. We propose that upregulation of a redox-active flavoprotein is a viable means to detoxify chromate that relies on adventitious reduction that is not catalyzed.

Dataset Information

Chromate Reductase YieF from Escherichia coli Enhances Hexavalent Chromium Resistance of Human HepG2 Cells.

Publications

Chromate Reductase YieF from Escherichia coli Enhances Hexavalent Chromium Resistance of Human HepG2 Cells.

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