Part:BBa_M45136

This part has a function to reduce uranium (VI) and chromate (VI) to more stable and insoluble form of uranium (IV) and chromate (III). It can also function in the reduction of some other heavy metals such plutonium and technetium.

E.Coli ChrR is an obligate two-electron reducer membrane-bound enzyme that is soluble and easy to manipulate. It reduces the chromate and uranium (uranyl) intracellularly from their unstable high valent states to stable and lower valent states. Since the reduction process takes place intracellularly the reduced product can be partially sequestered, resulting in minimization of the possibilities of reoxidation ans resolubilization of these nuclear wastes which happens in bacterial cell-surface-mediated reduction. Another striking point of this enzyme is that it isNAD(P)H oxidoreductase, meaning it is active under both aerobic and anaerobic conditions as long as cofactors are present in the environment. Additionally, this enzyme can be effective in the reduction various of substrates such as; quinines, potassium ferricyanide, Mo (VI), 2,6-dichloroindophenol, methylene blue, cytichrome c, mitomycin C, 5-aziri-dinyl-2,4-dinitrobenzamide and 17-allyl-amino-17-demethoxygeldanamycin. Thus, it can remediate additional contaminants as well as chromate and uranyl radioactive wastes.

As seen in figure 1 its crystal structure, solved at 2.2 A resolution, indicates that it belongs to the flavodoxin superfamily in which flavin mononucleotide (FMN) is anchored to the protein. ChrR was crystallized as tetramer, and then size exclusion chromatography was performed and showed that oligomeric form leads to chromate reduction. Within the tetramer, the dimers interact with a pair of two hydrogen bond networks, each involving Tyr128 and Glu146 of one dimer and Arg125 and Tyr85 of the other. Changes in each of these amino acids enhanced chromate reductase activity of the enzyme. Therefore, this network is significantly involved in chromate reduction process.

Figure 1. Structure of Chromate Reductase, ChrR

Barak et al. (2006) improved ChrR by changing a few amino acids (Val120Ala, Tyr128Asn, Thr160Asn and Glu175Leu) to ChrR6 enzyme for chromate and uranyl reduction as seen in Figure 2. When they alter each amino acid individually, they found Tyr128Asn amino acid change is the most effective one among others in terms of enzyme activity because when this amino acid change is not done the enzyme activity becomes less than ChrR. Thus, Tyr128Asn was responsible for improvement of enzyme for chromate and uranyl reduction.

Figure 2. Chromate reductase specific activities of crude extracts of recombinant E. coli cells expressing E. coli ChrR and ChrR6.

The same research group formed a lot of distinct versions of ChrR by changing different amino acids in 2008. They had a novel statistical approach to improve the enzyme in the absence of exact structural information. One of the modified ChrR enzymes, ChrR30, exhibits the highest chromate and uranyl reductase activity as seen Table 1 which shows the uranyl reduction kinetic data. For chromate reductase, the enzyme activity (Vmax) goes up from 295±27 to 458 300±83 600 for ChrR and ChrR30, respectively. The amino acid changes are Y128N, G150S and N154T. These amino acid changes give rise to sharp increase in uranyl and chromate reductase enzyme activity.

Table 1. Uranyl reduction kinetics of selected evolved mutants

As a conclusion, the modified version of ChrR enzyme, ChrR30, reduces more effectively uranium (VI) and chromate (VI) to uranium (IV) and chromate (III) valent states intracellularly. This enzyme has also potential activity of reducing other metals or radionuclides such as; plutonium and technetium.