Chromate-Reducing Properties of Soluble Flavoproteins from Pseudomonas putida and Escherichia coli

Cr(VI) (chromate) is a serious environmental pollutant due to the wide use of chromium compounds in industries such as tanning, corrosion control, plating, pigment manufacture, and nuclear weapons production (2). At the Department of Energy (DOE) waste sites, for example, it is the second most common heavy-metal contaminant, ranging in concentration between 0.008 and 173 μM in groundwater and 98 nM and 76 mM in soil and sediments (30); since soil water is stored in small capillary spaces, the last-mentioned concentrations are very high. Chromate is toxic, mutagenic, and carcinogenic (14, 35). Several factors contribute to its toxicity. Because of its structural similarity to SO42−, it is readily taken up by both bacterial and eukaryotic cells through the sulfate transport system (5, 35). Inside the cell, it is reduced nonenzymatically, as well as by various enzymes. Its partial reduction, particularly by the cellular one-electron reducers, generates Cr(V) and reactive oxygen species (ROS); the latter may be a major factor in causing cellular damage (5, 35, 39). Chromate is soluble and thus readily spreads beyond the site of initial contamination. Bacteria can convert chromate to Cr(III), which is much less toxic and less soluble, and thus bacterial bioremediation of chromate is of considerable interest, especially given the fact that alternative chemical means are prohibitively expensive for large-scale cleanup (5). Field studies have shown that biostimulation is a promising approach for bioremediation (19, 24). This method entails addition of nutrients to the environment, such as aquifers, to stimulate the growth of indigenous bacteria. Although enhanced growth does promote bioremediation, the resulting large amount of biomass can result in clogging of subsurface pores and confining of remediation to a narrow zone (19). Moreover, polluted environments, especially the DOE sites (30), contain multiple pollutants. Biostimulation of such sites is likely to have limited effectiveness, since the remediating bacteria, as well as the enzymes involved, are inhibited by the mixed waste present in such environments (M. Keyhan and A. Matin, unpublished data). Chromate remediation in such environments is further complicated by the fact that its reduction involves the generation of toxic intermediates, which are detrimental to the remediating bacteria (5). One way to address these problems is to use genetic- and protein engineering approaches. For instance, the use of appropriate promoters can ensure maximal expression of desired genes in slowly growing bacteria, thereby minimizing biomass formation and clogging (21, 22). Also, protein engineering of bacterial chromate reductases can generate improved enzymes that reduce chromate more efficiently, that minimize chromate toxicity to the remediating bacteria, and that can function in the presence of other pollutants. Some bacteria can evidently use chromate as the terminal electron acceptor, employing membrane-bound enzymes (23, 25, 41). However, several others reduce it using soluble enzymes (4, 12, 28, 39). The functional aspects of the latter group have been little studied, and they are more amenable to protein engineering. We have therefore focused on characterizing the soluble chromate reductases to identify suitable candidates for further work. Using classical biochemical techniques, a novel soluble enzyme (ChrR) with chromate reductase activity was previously purified to homogeneity from Pseudomonas putida (28). N-terminal and internal amino acid sequence determination of the enzyme allowed the design of appropriate primers to clone the chrR gene (27). BLAST searching of protein databases with the derived ChrR amino acid sequence revealed a conserved family of proteins whose members are present in a wide range of organisms. Over 40 of these homologs, including the predicted product of a previously uncharacterized open reading frame (yieF) from Escherichia coli, show >30% amino acid identity with ChrR. All contain the characteristic signature of the NADH_dh2 family of proteins, which consists of bacterial and eukaryotic NAD(P)H oxidoreductases (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi). Using appropriate primers, the E. coli yieF gene was cloned as well, and it was shown that this enzyme also can reduce chromate (27). The availability of the cloned genes has enabled us to obtain large quantities of the two enzymes in electrophoretically pure form and to compare several of their characteristics: chromate reduction kinetics, generation of ROS during chromate reduction, and induction patterns. While both enzymes are suitable for further work to improve bacterial chromate bioremediation, YieF appears to be a better candidate.