Vibrio harveyi nitroreductase is also a chromate reductase

Abstract

The chromate reductase purified from Pseudomonas ambigua was found to be homologous with several nitroreductases. Escherichia coli DH5alpha 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 DH5alpha NfsA (GST-EcNfsA), a fusion between GST and E. coli DH5alpha 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 micro 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 degrees 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).

FIG. 1.

Purification of nitroreductases fused to GST. E. coli DH5α (pGST-EcNfsA), E. coli DH5α (pGST-EcNfsB), and E. coli DH5α (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.

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 (▪), GST-EcNfsB (□), 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 (▪), 166 μg of GST-EcNfsB (□), 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.

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%.

FIG. 4.

Effect of temperature on chromate reductase activity. The basic enzyme reaction mixtures containing GST-EcNfsA (▪), GST-EcNfsB (□), 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.

FIG. 5.

NADH consumption. The reaction mixture contained 164 μg of enzyme GST-VhNfsA with 30 μM chromate (▪) or without chromate (□) 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 (•).