Cr(VI) (chromate) is a toxic, soluble environmental contaminant. Bacteria can reduce chromate to the insoluble and less toxic Cr(III), and thus chromate bioremediation is of interest. Genetic and protein engineering of suitable enzymes can improve bacterial bioremediation. Many bacterial enzymes catalyze one-electron reduction of chromate, generating Cr(V), which redox cycles, generating excessive reactive oxygen species (ROS). Such enzymes are not appropriate for bioremediation, as they harm the bacteria and their primary end product is not Cr(III). In this work, the chromate reductase activities of two electrophoretically pure soluble bacterial flavoproteins—ChrR (from Pseudomonas putida ) and YieF (from Escherichia coli )—were examined. Both are dimers and reduce chromate efficiently to Cr(III) ( k _cat / K _m = ∼2 × 10 ⁴ M ⁻¹ · s ⁻¹ ). The ChrR dimer generated a flavin semiquinone during chromate reduction and transferred >25% of the NADH electrons to ROS. However, the semiquinone was formed transiently and ROS diminished with time. Thus, ChrR probably generates Cr(V), but only transiently. Studies with mutants showed that ChrR protects against chromate toxicity; this is possibly because it preempts chromate reduction by the cellular one-electron reducers, thereby minimizing ROS generation. ChrR is thus a suitable enzyme for further studies. During chromate reduction by YieF, no flavin semiquinone was generated and only 25% of the NADH electrons were transferred to ROS. The YieF dimer may therefore be an obligatory four-electron chromate reducer which in one step transfers three electrons to chromate and one to molecular oxygen. As a mutant lacking this enzyme could not be obtained, the role of YieF in chromate protection could not be directly explored. The results nevertheless suggest that YieF may be an even more suitable candidate for further studies than ChrR.

Summary: Chromate [Cr(VI)] is a serious environmental pollutant, which is amenable to bacterial bioremediation. NfsA, the major oxygen‐insensitive nitroreductase of Escherichia coli , is a flavoprotein that is able to reduce chromate to less soluble and less toxic Cr(III). We show that this process involves single‐electron transfer, giving rise to a flavin semiquinone form of NfsA and Cr(V) as intermediates, which redox cycle, generating more reactive oxygen species (ROS) than a divalent chromate reducer, YieF. However, NfsA generates less ROS than a known one‐electron chromate reducer, lipoyl dehydrogenase (LpDH), suggesting that NfsA employs a mixture of uni‐ and di‐valent electron transfer steps. The presence of YieF, ChrR (another chromate reductase we previously characterized), or NfsA in an LpDH‐catalysed chromate reduction reaction decreased ROS generation by c . 65, 40, or 20%, respectively, suggesting that these enzymes can pre‐empt ROS generation by LpDH. We previously showed that ChrR protects Pseudomonas putida against chromate toxicity; here we show that NfsA or YieF overproduction can also increase the tolerance of E. coli to this compound.

Lichens, which are defined by a symbiosis between a mycobiont (fungal partner) and a photobiont (photoautotrophic partner), are in fact complex assemblages of microorganisms that constitute a largely untapped source of bioactive secondary metabolites.

The nature of the stress experienced by Escherichia coli K-12 exposed to chromate, and mechanisms that may enable cells to withstand this stress, were examined. Cells that had been preadapted by overnight growth in the presence of chromate were less stressed than nonadapted controls. Within 3 h of chromate exposure, the latter ceased growth and exhibited extreme filamentous morphology; by 5 h there was partial recovery with restoration of relatively normal cell morphology. In contrast, preadapted cells were less drastically affected in their morphology and growth. Cellular oxidative stress, as monitored by use of an H ₂ O ₂ -responsive fluorescent dye, was most severe in the nonadapted cells at 3 h postinoculation, lower in the partially recovered cells at 5 h postinoculation, and lower still in the preadapted cells. Chromate exposure depleted cellular levels of reduced glutathione and other free thiols to a greater extent in nonadapted than preadapted cells. In both cell types, the SOS response was activated, and levels of proteins such as SodB and CysK, which can counter oxidative stress, were increased. Some mutants missing antioxidant proteins (SodB, CysK, YieF, or KatE) were more sensitive to chromate. Thus, oxidative stress plays a major role in chromate toxicity in vivo, and cellular defense against this toxicity involves activation of antioxidant mechanisms. As bacterial chromate bioremediation is limited by the toxicity of chromate, minimizing oxidative stress during bacterial chromate reduction and bolstering the capacity of these organisms to deal with this stress will improve their effectiveness in chromate bioremediation.

Most bacteria contain soluble quinone-reducing flavoenzymes. However, no biological benefit for this activity has previously been demonstrated. ChrR of Pseudomonas putida is one such enzyme that has also been characterized as a chromate reductase; yet we propose that it is the quinone-reducing activity of ChrR that has the greatest biological significance. ChrR reduces quinones by simultaneous two-electron transfer, avoiding formation of highly reactive semiquinone intermediates and producing quinols that promote tolerance of H(2)O(2). Expression of chrR was induced by H(2)O(2), and levels of chrR expression in overexpressing, wild type, and knock-out mutant strains correlated with the H(2)O(2) tolerance and scavenging ability of each strain. The chrR expression level also correlated with intracellular H(2)O(2) levels as measured by protein carbonylation assays and fluorescence-activated cell scanning analysis with the H(2)O(2)-responsive dye H(2)DCFDA. Thus, enhancing the activity of ChrR in a chromate-remediating bacterial strain may not only increase the rate of chromate transformation, it may also augment the capacity of these cells to withstand the unavoidable production of H(2)O(2) that accompanies chromate reduction.

This review examines the vast catalytic and therapeutic potential offered by type I (i.e. oxygen-insensitive) nitroreductase enzymes in partnership with nitroaromatic prodrugs, with particular focus on gene-directed enzyme prodrug therapy (GDEPT; a form of cancer gene therapy). Important first indications of this potential were demonstrated over 20 years ago, for the enzyme–prodrug pairing of Escherichia coli NfsB and CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide]. However, it has become apparent that both the enzyme and the prodrug in this prototypical pairing have limitations that have impeded their clinical progression. Recently, substantial advances have been made in the biodiscovery and engineering of superior nitroreductase variants, in particular development of elegant high-throughput screening capabilities to enable optimization of desirable activities via directed evolution. These advances in enzymology have been paralleled by advances in medicinal chemistry, leading to the development of second- and third-generation nitroaromatic prodrugs that offer substantial advantages over CB1954 for nitroreductase GDEPT, including greater dose-potency and enhanced ability of the activated metabolite(s) to exhibit a local bystander effect. In addition to forging substantial progress towards future clinical trials, this research is supporting other fields, most notably the development and improvement of targeted cellular ablation capabilities in small animal models, such as zebrafish, to enable cell-specific physiology or regeneration studies.