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Applied and Environmental Microbiology, August 2003, p . 4390-4395, Vol . 69, No . 8

Vibrio harveyi Nitroreductase Is Also a Chromate Reductase

Young Hak Kwak,1 Dong Seok Lee,2 and Han Bok Kim1*

Department of Life Science, Hoseo University, Asan-Si, ChungNam 336-795,1 Department of Medical Laboratory Science, Inje University, Kimhae 621-749, Korea2

Received 1 October 2002/ Accepted 14 May 2003


   ABSTRACT

 
The chromate reductase purified from Pseudomonas ambigua was found to be homologous with several nitroreductases . Escherichia coli DH5{alpha} and Vibrio harveyi KCTC 2720 nitroreductases were chosen for the present study, and their chromate-reducing activities were determined . A fusion between glutathione S-transferase (GST) and E . coli DH5{alpha} NfsA (GST-EcNfsA), a fusion between GST and E . coli DH5{alpha} 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 Km values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 11.8, 23.5, and 5.4 µM, respectively . The Vmax 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 Vmax/Km 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) .


   INTRODUCTION

 
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, 33-35) . 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%) and Vibrio harveyi NfsA (58%) nitroreductase .

There are two types of nitroreductase . 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) . NfsA includes the major nitroreductase in E . coli (3, 37) and Frp in V . harveyi (14, 17) . NfsB includes the minor nitroreductase in E . coli (3, 20, 38) and Frase I in Vibrio (Photobacterium) fischeri (6, 39) .

The E . coli and V . harveyi nitroreductases were chosen for the present study . It will be shown that these nitroreductases are also efficient in chromate reduction .


   MATERIALS AND METHODS

 
Bacterial strains and plasmids.
E . coli DH5{alpha} was grown on Luria-Bertani (LB) broth for 12 h at 37°C . Freeze-dried V . harveyi KCTC 2720 cells were resuspended in sterile distilled water . The cell suspension was directly used in PCR . V . harveyi KCTC 2720 was from the Korean Collection for Type Cultures (KCTC), Seoul, Korea .

Cloning of nitroreductase gene.
To clone the nfsA gene of E . coli DH5{alpha}, two primers (EcNfsA1 [5'-GTAGGATCCACGCCAACCATTGAAC-3'] containing a BamHI site, and EcNfsA2 [5'-ACTGAATTCTTAGCGCGTCGCCCAAC-3'] containing a EcoRI site) were used . The two primer sequences were deduced from E . coli AB1157 (36) . To clone the nfsB gene from E . coli DH5{alpha}, two primers (EcNfsB1 [5'-GTAGGATCCGATATCATTTCTGTCGC-3'] containing a BamHI site and EcNfsB2 [5'-ACTGAATTCTTACACTTCGGTTAAGGTG-3'] containing a EcoRI site) were used . The two primer sequences were deduced from E . coli C600 (38) . To clone the nfsA gene from V . harveyi KCTC 2720, two primers (VhNfsA1 [5'-GTAGGATCCAACAATACGATTGAAAC-3'] containing a BamHI site and VhNfsA2 [5'-ACTGAATTCTTAGCGTTTTGCTAGCC-3'] containing a EcoRI site) were used . The two primer sequences were from V . harveyi ATCC 33843 (17) . PCRs were carried out in a 50-µl reaction volume . A 1-µl volume of suspended cells was added to 49 µl of PCR mixture (50 mM KCl; 10 mM Tris-HCl [pH 9.0]; 0.1% Triton X-100; 1.5 mM MgCl2; 0.25 mM [each] dATP, dGTP, dCTP, and dTTP [Sigma, St . Louis, Mo.]; 10 pmol (0.2 x 10-3 mM] of each primer, and 2.5 U of Taq DNA polymerase [Promega Co., Madison, Wis.]) . PCR amplification was performed in a Perkin-Elmer thermal cycler with the following steps: an initial denaturation step at 95°C for 7 min; 30 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min; and a final extension step of 72°C for 5 min . Amplified DNA fragments were electrophoresed in a 1.5% agarose gel and were eluted by using a GeneClean II kit (Bio 101, Inc., La Jolla, Calif.) . For easy purification of nitroreductase, the three nitroreductase genes were fused into the glutathione S-transferase (GST) gene (32) . Each PCR product was cut with BamHI and EcoRI (Promega Corp., Madison, Wis.), electrophoresed in a 1.5% agarose gel, and eluted with the GeneClean II kit . It was ligated by T4 DNA ligase (TaKaRa Co., Kyoto, Japan) into plasmid pGEX-4T-1 (Pharmacia Biotech) containing GST that was also cut with BamHI and EcoRI . E . coli BL21 cells were transformed with the ligation mixture . The strain containing the fusion genes on the plasmids was screened by ampicillin resistance and plasmid digestion with the two enzymes . GST-EcNfsA is a fusion between GST at the N terminus and upstream from E . coli DH5{alpha} NfsA, and GST-EcNfsB is a fusion between GST and E . coli DH5{alpha} NfsB . GST-VhNfsA is a fusion between GST and V . harveyi KCTC2720 NfsA . All three nitroreductase fusion genes on the plasmid (pGST-EcNfsA, pGST-EcNfsB, and pGST-VhNfsA) were confirmed by one primer in the nitroreductase gene (EcNfsA1, EcNfsB1, and VhNfsA1) and the other on the plasmid (CR2) . The sequence of CR2 is 5'-GGGAGCTGCATGTGTCAGAG-3' .

DNA sequencing and sequence analysis.
All three nitroreductase genes were obtained from pGST-EcNfsA, pGST-EcNfsB, and pGST-VhNfsA and were then subcloned into pUC18 . Their nucleotide sequences were determined by using ThermoSequenase (Amersham Life Science, Arlington Heights, Ill.) and the dideoxy termination method . The accession numbers for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA deposited in GenBank are AF394662, AF394661, and AF395832, respectively .

Purification of fused nitroreductase.
E . coli BL21 harboring the plasmids containing fused nitroreductase genes was cultured at 37°C for 14 h and diluted 100-fold in fresh LB broth . It was cultured again to reach its exponential phase . In this phase, protein expression was induced by adding 0.5 mM IPTG (isopropyl-ß-D-thiogalactopyranoside) for 3.5 h . The cells were harvested by centrifugation at 13,000 x g for 5 min . To purify the fused nitroreductase, affinity chromatography was used . Cell extracts were obtained by sonication . The extracts and glutathione-agarose beads (Sigma) were mixed . The fused nitroreductase bound to the glutathione-agarose beads was eluted with 10 mM reduced glutathione (pH 8.0) . The protein concentration was determined either by measuring the optical density values at A280 or by the Bradford method with a Serva dye and with bovine serum albumin as a standard (28) .

SDS-PAGE.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on a 12.5% gel by the method of Laemmli (16) . Protein bands were visualized with Coomassie brilliant blue R-250 (Sigma) .

Gel filtration.
Gel filtration was carried out in a 2.5-by-50-cm column of Sephadex G-75 (Sigma) . The Sephadex G-75 column was equilibrated in 20 mM Tris-HCl (pH 7.0) buffer at a flow rate of 0.6 ml/min . The void volume (Vo) was determined by using blue dextran (Sigma) . The standard proteins (Sigma) used for the estimation of molecular mass included bovine albumin (66 kDa), egg albumin (45 kDa), rabbit glyceraldehyde-3-phosphate dehydrogenase (36 kDa), bovine carbonic anhydrase, (29 kDa), and bovine trypsinogen (24 kDa) . The elution profiles were monitored by UV absorption at 280 nm . SDS-PAGE was used to characterize the sizes and types of subunits in the purified chromate reductases .

Spectral measurements.
The absorption spectrum of nitroreductase was measured with spectrophotometer (UVICON930) in a range between 250 and 500 nm .

Identification of the nitroreductase-bound flavin.
To characterize the cofactor in the nitroreductases, the purified nitroreductases were boiled and precipitated to extract the cofactor . After centrifugation, the supernatant (20 µl) containing the cofactor was spotted and chromatographed in the dark on silica gel 60 F254 (Merck) with FAD and FMN standards by using a solvent system (distilled water-ethanol-acetic acid [20:6:1]) . The position of each spot was visualized by using 365-nm UV .

Determination of nitroreductase activity.
Nitrofurazone nitroreductase activity was assayed in a reaction mixture containing 10 µM Tris-HCl (pH 7.5), 10 µM nitrofurazone (Sigma), 0.1 mM NADH (Sigma), and GST-EcNfsA, GST-EcNfsB, or GST-VhNfsA enzyme . Nitroreductase activity was determined by a decrease in absorbance at 400 nm (36) . The transformation of TNT was indicated by a decrease in the absorbance of TNT at 447 nm (22) . The assay mixture contained 50 mM Tris-HCl (pH 7.0), 0.1 mM TNT, 1 mM NADH, and either GST-EcNfsA, GST-EcNfsB, or GST-VhNfsA enzyme .

Determination of chromate reductase activity.
A standard curve for the reaction between chromate and its binding dye, 1,5-diphenylcarbazide (Sigma), was used to determine the chromate concentration (13) . The diphenylcarbazide forms a pink complex with Cr(VI) but not with Cr(III) . Reduction of Cr(VI) can produce Cr(V) and Cr(IV) . However, Cr(V) and Cr(IV) are not stable in aqueous states at neutral pH (25) . Thus, it is valid to measure Cr(VI) conversion to Cr(III) by using the dye method .

The chromate reductase assay was performed in 10 mM Tris-HCl (pH 7.0) buffer containing 1 mM NADH, 1 mM K2CrO4 (Sigma), and the enzyme at 30°C for 1 h . Then, 10 µl of 0.1 M H2SO4 and 15 µl of 0.5% (vol/vol) 1,5-diphenyl carbazide were added to the tube, and the residual Cr(VI) content was determined at 540 nm . The optimal temperature of chromate reductase activity was determined at various temperatures ranging from 4 to 80°C .

Determination of chromate reductase kinetics.
Various concentrations of K2CrO4 were added to the basic enzyme reaction mixture . Km and Vmax values were determined by using a Lineweaver-Burk plot .


   RESULTS

 
Cloning of nitroreductase genes.
The chromate reductase from P . ambigua (33) was compared to the E . coli and P . ambigua nitroreductases . Surprisingly, a high degree of homology between P . ambigua chromate reductase and E . coli nitroreductase NfsA (59%) and between P . ambigua reductase and V . harveyi nitroreductase NfsA (58%) was found, as determined by using the BLAST function of the National Center for Biotechnology Information . It was of interest to determine whether the E . coli NfsA and NfsB and the V . harveyi NfsA nitroreductases had chromate reductase activities similar to that of the P . ambigua chromate reductase .

The PCR products of E . coli DH5{alpha} nfsA and nfsB and of V . harveyi KCTC 2720 nfsA nitroreductase genes consisted of 738, 669, and 738 bp, respectively, just as predicted (data not shown) .

The three nitroreductase genes were sequenced . No differences were found between the E . coli DH5{alpha} and AB1157 nfsA genes and the E . coli DH5{alpha} and C600 nfsB genes (GenBank numbers AF394662 and AF394661, respectively) . However, eight nucleotide sequences in the nfsA nitroreductase genes were different between the two V . harveyi strains (GenBank no . AF395832) . Five amino acid residues in the nitroreductase in V . harveyi ATCC 33843 were replaced by different residues in V . harveyi KCTC 2720 (data not shown) .

Some biochemical characteristics of fused nitroreductase.
Using the affinity between glutathione and its S-transferase, the three fused nitroreductases were purified to homogeneity and used in biochemical studies (Fig . 1) . The molecular masses of GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA were each determined to be 50 kDa on an SDS-PAGE gel (Fig . 1) . The absorbance of GST-EcNfsA (GST-EcNfsB and GST-VhNfsA) was maximal at 370 and 450 nm (data not shown) . This suggests that GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA are typical flavoproteins containing FAD or FMN . Thin-layer chromatography analysis showed that GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA contain FMN rather than FAD (data not shown) . GST used as a control did not contain FMN at all (data not shown) .


FIG . 1 . Purification of nitroreductases fused to GST . E . coli DH5{alpha} (pGST-EcNfsA), E . coli DH5{alpha} (pGST-EcNfsB), and E . coli DH5{alpha} (pGST-VhNfsA) were cultured to exponential phase in LB broth and then induced by the addition of 0.5 mM IPTG for 3.5 h . Induced cells were broken by sonication and separated into supernatant and pellet fractions by centrifugation . By using affinity chromatography of glutathione and GST, GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA (lanes 3, 4, and 5, respectively) were purified . Lanes 2 and 6 show GST (26 kDa) and BSA (66 kDa), respectively . Lane 1 has molecular-mass standards.

 
Nitroreductase activity.
We determined whether a fusion of GST and nitroreductase still contains nitroreductase activity . Substrates for E . coli nitroreductase are menadione (vitamin K), nitrofurazone, TNT, etc . (7, 11, 19, 22, 36) . We chose nitrofurazone and TNT as the substrates for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA . We postulated that electrons move from NADH to substrate nitrofurazone or TNT in the presence of nitroreductase . We found that GST-EcNfsB was the most efficient in nitrofurazone-reducing activity and that GST-VhNfsA was as efficient as GST-EcNfsA (Fig . 2A) . GST-VhNfsA was most efficient in TNT reduction, GST-EcNfsB is next most efficient, and GST-EcNfsA is least efficient (Fig . 2B) . These results demonstrated clearly that all three—GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA—retained nitroreductase activities .


FIG . 2 . (A) Nitrofurazone-reducing activity . The reduction of nitrofurazone was determined by measuring the decrease in the absorbance of nitrofurazone at 400 nm (molar extinction coefficient, 12,960 M-1 cm-1) . A basic enzyme reaction mixture (1.5 ml) containing 10 mM Tris-HCl buffer (pH 7.0), 0.1 mM NADH, 10 µM nitrofurazone, and 10 µg of either GST-EcNfsA ({blacksquare}), GST-EcNfsB ({square}), or GST-VhNfsA (•) was incubated at 30°C . The reaction was started by the addition of NADH . One unit was defined as the amount of the enzyme that reduced 1 pmol of nitrofurazone/µg of protein . (B) TNT-reducing activity . The transformation of TNT was determined by measuring the decrease in the absorbance of TNT at 447 nm . The standard assay mixture (1 ml) containing 50 mM Tris-HCl (pH 7.0), 0.1 mM TNT, 1 mM NADH, and either 172 µg of GST-EcNfsA ({blacksquare}), 166 µg of GST-EcNfsB ({square}), or 82 µg of GST-VhNfsA (•) was incubated at 30°C . Theenzymatic reaction was initiated by adding NADH . The reaction was quenched by adding 160 µl of 1 M NaOH, resulting in a pH of 12.2 . Quantitative measurements were made 5 min after the addition of NADH to the TNT solution . One unit was defined as the amount of the enzyme that reduced 1 pmol of TNT/µg of protein.

 
The molecular masses of GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA were each also determined to be 50 kDa by gel filtration (data not shown) . They were 50 kDa on SDS-PAGE (Fig . 1) . Thus, purified GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA are monomers in solution . In contrast, P . ambigua chromate reductase is a dimer (33) .

Chromate reductase activity.
We determined whether the fused nitroreductase had chromate reductase activity like P . ambigua chromate reductase .

Glutathione (0.2 mM) is known to have chromate-reducing activity (33) . However, 0.025 mM glutathione, which was present in the assay buffer, did not demonstrate any chromate-reducing activity . The GST used in the present study did not retain any chromate-reducing activity (data not shown) .

NADH alone showed chromate-reducing activity (Fig . 3) . However, when chromate reductase was added to the reaction mixture, the chromate-reducing activities of GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA increased by 2.6, 2.6, and 4.2 fold, respectively (Fig . 3) . Thus, GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA are real chromate reductases in the presence of coenzyme NADH . When the NADH concentration in the reaction mixture was increased from 0.05 to 1 mM, the chromate reductase activities of GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA increased (Fig . 3) . This confirms again that GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA require NADH for their activities . P . ambigua chromate reductase is also NADH dependent (33) . NADPH was almost as efficient as NADH in chromate reduction by GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA (data not shown) .


FIG . 3 . NADH-dependent chromate reductase activity . Three kinds of reaction mixtures were prepared: 10 mM Tris-HCl (pH 7.0), 0.1 mM NADH, 20 µM K2CrO4, and either GST-EcNfsA, GST-EcNfsB, or GST-VhNfsA (); 10 mM Tris-HCl (pH 7.0), 0.1 mM NADH, 20 µM K2CrO4 (no enzyme) (); and 10 mM Tris-HCl (pH 7.0), 0.05 mM NADH, 20 µM K2CrO4, and either GST-EcNfsA, GST-EcNfsB, or GST-VhNfsA () . They were incubated at 30°C for 1 h, and the chromate reductase activity was determined by the dye method described in the text . One unit of chromate reductase activity was defined as the amount of the enzyme which decreased 1 nmol of Cr(VI) per min at 30°C per mg of protein . The chromate reductase activity of GST-VhNfsA in the reaction buffer () was regarded as 100%.

 
Activity of GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA was optimal at 55, 30, and 30°C, respectively (Fig . 4) . GST-EcNfsB is characteristic of being still active at 4°C, retaining 65% of its maximum activity . GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA all lost considerable activities at >60°C (Fig . 4) .


FIG . 4 . Effect of temperature on chromate reductase activity . The basic enzyme reaction mixtures containing GST-EcNfsA ({blacksquare}), GST-EcNfsB ({square}), or GST-VhNfsA (•) were incubated at 4 to 80°C, and their chromate-reducing activities were determined . The chromate reductase activity of GST-EcNfsA at 55°C, GST-EcNfsB at 30°C, or GST-VhNfsA at 30°C was regarded as the 100% value.

 
All three nitroreductases showed chromate reductase activity (Table 1) . Vmax/Km value of each nitroreductase can be used as a measure of the efficiency of the chromate reductase activity . GST-VhNfsA was most efficient, and GST-EcNfsA was twice as efficient as GST-EcNfsB (Table 1) . The chromate reductase activity of GST-VhNfsA (12 times as efficient as GST-EcNfsB) matched that of P . ambigua chromate reductase (13 times as efficient as GST-EcNfsB) (Table 1) (33) . This confirms that the nitroreductases are effective in chromate reduction .


TABLE 1 . Comparison of Cr(VI) reductase kinetics

 
Cr(III) production from Cr(VI) (chromate) by GST-VhNfsA was confirmed by the appearance of an absorption peak at 567 nm, as determined by Puzon et al . (27; data not shown) . A typical Cr(III) greenish color was also observed in chromate reduction by GST-VhNfsA . Surprisingly, Cr(III) produced by GST-VhNfsA enzyme was soluble, which is consistent with data of Puzon et al . (27) . CrCl3 (5 mM) in pH 7 solution was insoluble (data not shown) . Although Cr(III) produced by GST-VhNfsA was soluble, Cr(III) toxicity was reduced considerably, since it did not inhibit growth of E . coli (data not shown) .

NADH consumption.
In the presence of enzyme GST-VhNfsA, NADH is oxidized even without chromate (Fig . 5) . When chromate was added into the reaction buffer containing NADH and enzyme GST-VhNfsA, NADH consumption rate increased (Fig . 5), suggesting that electrons move from NADH onto Cr(VI) . The ratio between the amount of NADH consumed for chromate reduction and that of reduced chromate was approximately 3 (Fig . 5) .


FIG . 5 . NADH consumption . The reaction mixture contained 164 µg of enzyme GST-VhNfsA with 30 µM chromate ({blacksquare}) or without chromate ({square}) in 1 ml of 10 mM Tris-HCl (pH 7.0), and NADH (final concentration 100 µM) was then added . The absorbance change was determined at 340 nm after every 1-min interval . The amount of reduced chromate in the reaction buffer containing 30 µM chromate, 100 µM NADH, and GST-VhNfsA was determined by the dye method described in the text at 5-min intervals (•).

 

   DISCUSSION

 
We selected E . coli and V . harveyi nitroreductases and determined their chromate reductase activities, since P . ambigua chromate reductase was homologous with them . We did find that the nitroreductases are effective in chromate reduction . To our knowledge, we are the first to recognize that nitroreductase can also act as a chromate reductase .

The chromate-reducing activity of GST-VhNfsA was 12 times as efficient as that of GST-EcNfsB, and that of GST-EcNfsA is twice as efficient as that of GST-EcNfsB (Table 1), suggesting that the nfsA(GST-VhNfsA, GST-EcNfsA) family is more efficient than nfsB(GST-EcNfsB) as a chromate reductase .

GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA showed optimum activities at physiological temperatures (20 to 40°C) (Fig . 4) . Most of the enzymes were inactive at 4°C . However, GST-EcNfsB still showed 60% of its maximum activity at 4°C (Fig . 4) . This property might be exploited when the enzyme is used under in situ bioremediation conditions even in winter .

P . putida chromate reductase has a high Vmax value of 1.72 µmol/min/mg of protein (24) . However, its Km value of 374 µM (24) is 70 times higher than that of GST-VhNfsA . Overall, the chromate reductase efficiency of P . putida is similar to that of GST-VhNfsA . GST-VhNfsA activity was optimal at 30°C, whereas P . putida chromate reductase efficiency is optimal at 80°C . In actual bioremediation, GST-VhNfsA activity is expected to be more useful than that of P . putida chromate reductase. Puzon et al . (27) reported on a bacterial flavin reductase, Fre, that reduces chromate . It exhibits a very high Vmax value . Since these authors did not report its Km value, GST-VhNfsA cannot be compared to it . Fre did not contain a bound flavin and needed free FMN for its chromate reduction (27), whereas GST-VhNfsA contained a bound FMN .

Cr(III) is assumed to precipitate as Cr(OH)3 or Cr2O3 in neutral solution (27) . Chromate remediation has been based on the fact that Cr(III) is less soluble and less toxic . However, we found that the Cr(III) produced by GST-VhNfsA was soluble . Puzon et al . (27) reported that Cr(III)-NAD+ complex produced by Fre enzyme was also soluble . The Cr(III) produced by GST-VhNfsA might have the similar structure . The fact that the Cr(III) produced by enzyme is mobile was disappointing at first . However, the Cr(III) produced by GST-VhNfsA was much less toxic to E . coli, and this is a favorable aspect in chromate remediation .

Some reductase in the cell might have diverse functions, including a chromate-reducing activity . It was demonstrated here that some E . coli and V . harveyi nitroreductases are also efficient in reducing chromate . The reductase or operons for copper, arsenite, and mercury are known (31) . However, the operon for chromate is not yet known .

 


   FOOTNOTES

 
* Corresponding author . Mailing address: Department of Life Science, Hoseo University, Asan-Si, ChungNam 336-795, Korea . Phone: (82) 41-540-5624 . Fax: (82) 41-548-6231 . E-mail: hbkim{at}office.hoseo.ac.kr .


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