Evaluation of Class II Chromate Reductases and their Bioremediation Potential

Microbial bio-reduction to Cr(III) is a promising strategy for detoxification of chromate, a prevalent anthropogenic pollutant. We propose that this activity can be enhanced in bacteria through engineering of soluble enzymes, such as NfsA, the model Class II chromate reductase of Escherichia coli. We show here that nfsA is induced by chromate, and that it reduces this compound by a “semi-tight” reaction mechanism, involving asynchronous transfer of electrons from its FMN cofactor to chromium. This mechanism produces a reactive Cr(V) intermediate; and consequently generates higher levels of reactive oxygen species (ROS) than the “tight” (simultaneous two-electron transferring) Class I chromate reducer YieF. Strengthening the activity of NfsA and other Class II enzymes by directed evolution would likely enhance chromate transformation by a host cell; but our data to date indicate that the Class I enzymes are superior candidates for such studies. INTRODUCTION. Hexavalent chromium [Cr(VI); most commonly chromate] is a widespread environmental pollutant, arising as a by-product of numerous industrial processes. Chromate is highly toxic, mutagenic and carcinogenic, and because of its solubility in water, chromate contamination can be difficult to contain (Singh et al, 1998). Bacteria can reduce Cr(VI) to Cr(III), offering promise for an environmentally friendly and affordable solution to chromate pollution. However, in situ remediation is limited by the toxicity of chromate and co-contaminants to remediating cells, and chromate has been projected to persist at dangerous levels at waste sites for over 1000 years (Okrent and Xing, 1993). To provide a potential solution to this problem we have proposed molecular engineering of cells and enzymes to decrease chromate toxicity to bacteria and increase levels of chromate transformation (Keyhan et al, 2004). Identification of suitable soluble chromate-reducing enzymes will enable directed evolution studies aimed at enhancing the activity of these enzymes. Previously we have described the biochemical purification, cloning and characterization of ChrR, a class I chromate-reducing flavoenzyme from Pseudomonas putida (Park et al, 2000; Ackerley et al, 2004a). Shortly after this work was published, Okazaki and coworkers released the sequence of a further chromate-reducing flavoenzyme from Pseudomonas ambigua that they had earlier characterized (Suzuki et al, 1992). These two FMN-binding enzymes share no homology with one another, but each belongs to a family that is highly conserved in bacteria and has homologs with greater than 30% sequence identity in over 60 genome-sequenced strains. We have cloned and characterized three homologs of each enzyme, and to distinguish the two families we refer to the ChrR homologs as Class I enzymes, and the P. ambigua enzyme 1 -----------------------MTPTIELICGHRSIRHFTDEPISEAQREAIINSARAT 37 2 -----------------------MNEVIKSLTDHRSIRSYTDEPVAQEQLDQIIEAVQSA 37 3 -------------------------MVKELLRNHSSVRIYDGNPISKEIIEELIATAQMA 35 4 MSLQDEALKAWQARYGEPANLPAADTVIAQMLQHRSVRAYSDLPVDEQMLSWAIAAAQSA 60 . : * *:* : . *: : . * :. : : 1 SSSSFLQCSSIIRITDKALREELVTLTGGQKHVAQAAEFWVFCADFNR---HLQICPDAQ 94 2 PSSINGQQVTVITVQDKERKKKISELAGGQPWIDQAPVFLLFCADFNRAKIALEDLHDFK 97 3 ATSHFVQAYSVIWVTDEEKKEKLGMLSGNPRQYETSGGAFVFCVDFKR----LQSAGKLE 91 4 STSSNLQAWSVLAVRDRERLARLARLSGNQRHVEQAPLFLVWLVDWSR---LRRLARTLQ 117 . :* * ::: : *. : *:*. : :: . *:. * . : 1 -LGLA----EQLLLGVVDTAMMAQNALIAAESLGLGGVYIGGLRNNIEAVTKLLKLPQHV 149 2 -MEITNG-LESVLVGAVDAGIALGTATAAAESLGLGTVPIGAVRGNPQELIELLELPKYV 155 3 GVDIVADSAENVLVGVADVSLFAQNFVVAAESMGYGICYIGGVRNKPEEISELFNLPEYV 151 4 APTAGIDYLESYTVGVVDAALAAQNAALAFEAQGLGIVYIGGMRNHPEAMSEELGLPNDT 177 *. :*. *. : . * *: * * **. :*. : : : : : **: . 1 LPLFGLCLGWP--ADNPDLKPRLPASILVHENSYQPLD--KGALAQYDEQLAEYYLTRGS 205 2 FPLSGLVIGHP--ADRSAKKPRLPQEAVNHQETYLNQDELTSHIQAYDEQMSEYMNKRTN 213 3 FPLFGLTIGVP--ARRNEVKPRLPVAAVLHENEYNTEK-YEELLPAYNDTMEAYYNNRSS 208 4 FAVFGMCVGHPDPAQPAEIKPRLAQSVVLHRERYEATEAEAVSVAAYDRRMSDFQHRQQR 237 :. : *: :* * * ****. : *. : * . : *: : : : α-8 α-9 β-5 β-6 α-7 α-5 β-4 α-6 β-2 α-3 β-3 α-4 α-1 β-1 α-2 1 NNR--RDTWSDHIRRTIIKESRPFILDYLHKQGWATR240 2 GKE--TRNWSQSIASYYERLYYPHIREMLEKQGFKVEK 249 3 NRK--IDNWTKQMADFLIEQRRPHIKDFLAKKGFNWK243 4 ENRSWSSQAVERVKGADSLSGRHRLRDALNTLGFGLR274