Four arsenic-resistance genes(arsB,arsC,arsH,arsR) have been discovered in Acidithiobacillus ferrooxidans.Their gene sequences have been identified and three different arsenic-resistance mechanisms have been elucidate...Four arsenic-resistance genes(arsB,arsC,arsH,arsR) have been discovered in Acidithiobacillus ferrooxidans.Their gene sequences have been identified and three different arsenic-resistance mechanisms have been elucidated.However,the function of the arsH gene in At.ferrooxidans remains unclear.In order to evaluate the function of the arsH gene,we cloned it and expressed it in Escherichia coli.The protein was purified and its relative molecular mass was determined by SDS-PAGE(Sodium dodecyl sulfate-polyacrylamide gel electrophoresis).The results indicated that the relative molecular mass of the purified ArsH was approximately 29 kDa.The purified protein ArsH from E.coli BL21 was a flavoprotein that oxidized in vitro NADPH with an optimal pH of 6.4.展开更多
AzoR is a homodimeric,flavin mononucleotide（FMN）-containing,NADH-dependent azoreductase from Escherichia coli.In this paper,we investigated the effect of the concentration of both AzoR and R59 G on the spectral beha...AzoR is a homodimeric,flavin mononucleotide（FMN）-containing,NADH-dependent azoreductase from Escherichia coli.In this paper,we investigated the effect of the concentration of both AzoR and R59 G on the spectral behavior of the bound FMN using two-dimensional fluorescence correlation spectra.Two cross peaks（530,490） and（580,530） were observed from the dilution-induced 2D asynchronous correlation map of wt AzoR,while only one cross peak appeared at（600,530） for R59 C mutant.This result indicated that the mutation at site 59 influenced the formation of dilution-induced intermediates.The specific activity of both AzoR and R59 G mutant was unaffected by dilution when the enzyme concentration is below 1 μmol/L,which suggested that no significant dissociation of FMN occurred at low concentrations.Additionally,in order to explore the origin of these intermediates,we carried out a2 D correlation analysis using excitation wavelength-dependent fluorescence emission spectroscopy.The results showed that there coexisted two types of FMN that emitted fluorescence at 530 nm and 500 nm,respectively.Taken together,these results suggested that the 2D method is a very powerful method to identify the heterogeneous distribution of the bound FMN in solution.展开更多
基金Project(50621063) supported by the National Natural Science Foundation of ChinaProject(2004CB619201) supported by the National Basic Research Program of China
文摘Four arsenic-resistance genes(arsB,arsC,arsH,arsR) have been discovered in Acidithiobacillus ferrooxidans.Their gene sequences have been identified and three different arsenic-resistance mechanisms have been elucidated.However,the function of the arsH gene in At.ferrooxidans remains unclear.In order to evaluate the function of the arsH gene,we cloned it and expressed it in Escherichia coli.The protein was purified and its relative molecular mass was determined by SDS-PAGE(Sodium dodecyl sulfate-polyacrylamide gel electrophoresis).The results indicated that the relative molecular mass of the purified ArsH was approximately 29 kDa.The purified protein ArsH from E.coli BL21 was a flavoprotein that oxidized in vitro NADPH with an optimal pH of 6.4.
基金fund by sub-project of National Science and Technology Major Project on Water Pollution Prevention and Control(No.2012ZX07203-003-Z04)supported by the Fundamental Research Funds for the Central Universities(No. ZYGX2012J112)
文摘AzoR is a homodimeric,flavin mononucleotide（FMN）-containing,NADH-dependent azoreductase from Escherichia coli.In this paper,we investigated the effect of the concentration of both AzoR and R59 G on the spectral behavior of the bound FMN using two-dimensional fluorescence correlation spectra.Two cross peaks（530,490） and（580,530） were observed from the dilution-induced 2D asynchronous correlation map of wt AzoR,while only one cross peak appeared at（600,530） for R59 C mutant.This result indicated that the mutation at site 59 influenced the formation of dilution-induced intermediates.The specific activity of both AzoR and R59 G mutant was unaffected by dilution when the enzyme concentration is below 1 μmol/L,which suggested that no significant dissociation of FMN occurred at low concentrations.Additionally,in order to explore the origin of these intermediates,we carried out a2 D correlation analysis using excitation wavelength-dependent fluorescence emission spectroscopy.The results showed that there coexisted two types of FMN that emitted fluorescence at 530 nm and 500 nm,respectively.Taken together,these results suggested that the 2D method is a very powerful method to identify the heterogeneous distribution of the bound FMN in solution.
