The mechanism of chromate reduction by Thermus scotoductus SA-01

[摘要] English: Recent contamination of the environment with Cr(VI) through various industrial activities is becoming an increasing problem to which microbial Cr(VI) reduction could prove an important alternative solution compared to currently available chemical and physical treatment options. Cr(VI) is highly toxic and has been shown to be a mutagen and carcinogen, whereas Cr(III) is considered relatively innocuous. A variety of Cr(VI)-resistant and reducing bacteria have been isolated from diverse environments both contaminated and uncontaminated with Cr(VI). In 1999, Kieft and co-workers isolated a Thermus scotoductus strain from groundwater of a South African gold mine at a depth of 3.2 km. This thermophilic bacteria was shown to be able to readily reduce a variety of metals including Fe(III), Mn(IV), Co(III)-EDTA, U(VI) and Cr(VI). This ubiquitous nature of Cr(VI) reducing bacteria and the fact that Cr(VI) rarely occurs naturally in the environment, has led several researches to proposed that these chromate reducing properties is the serendipitous activity of enzymes with other primary physiological functions. Thermus scotoductus SA-01 has the ability to tolerate up to 0.5 mM Cr(VI) during growth in a complex organic medium and reduce Cr(VI) under growth and non-growth conditions. The rate of chromate reduction is dependent on pH, temperature, initial Cr(VI) concentration and is abolished by metabolic inhibiters. Chromate reduction was catalyzed by cellular extracts using NADH as an electron donor indicating the chromate reduction mechanisms to be enzymatic. Subcellular fractionation studies indicated that Thermus scotoductus SA-01 possesses more than one chromate reduction mechanisms. A novel cytoplasmic chromate reductase was purified to homogeneity and shown to couple the oxidation of NAD(P)H to the reduction of Cr(VI). This homodimeric protein consisted of monomers of approximately 36 kDa with a non-covalently bound FMN and required the divalent metal Ca2+ for activity. The enzyme was optimally active at 65°C and a pH of 6.3, reducing 2 mol of NAD(P)H per mol Cr(VI), suggesting a mixed one- and two-electron transfer mechanism. N-terminal sequencing and screening of a genomic library of T. scotoductus SA-01 using a DIG-labelled DNA probe, revealed the cytoplasmic chromate reductase to be encoded for by an ORF of 1050 bp under the regulation of an E. coli σ70-like promoter. Sequence analysis showed the chromate reductase to be related to the Old Yellow Enzyme (OYE) family and in particular some xenobiotic reductases. The membrane-associated chromate reductase was also purified to homogeneity and shown to be distinct from the above mentioned cytoplasmic chromate reductase. The membrane-assocated reductase appears to be peripherally-associated with the membrane of T. scotoductus and consists of two identical subunits of approximately 48 kDa. The chromate reductase contained a non-covalently bound FAD co-factor and was optimally active at 65°C and a pH of 6.5. Through N-terminal sequencing and screening of a genomic library, the membrane-associated chromate reductase was identified as homologous to the dihydrolipoamide dehydrogenase gene, encoded for by a 1386 bp ORF and located within a probable pyruvate dehydrogenase operon. Although neither of these enzymes are dedicated physiological chromate reductases, their catalytic efficiency toward Cr(VI) as substrate proved to be superior than that found for other chromate reductases described in literature, which include the nitroreductases and quinone reductases isolated from Pseudomonas putida and Escherichia coli Both the cytoplasmic and membrane-associated chromate reductases were heterologously overproduced in E. coli and T. thermophilus as active, soluble enzymes. Kinetic studies of the recombinant proteins showed that the recombinant chromate reductases expressed in T. thermophilus were more catalytic efficient than their E. coli-expressed counterparts. Although structurally no differences could be observed using UV-vis and circular dichroism spectroscopy, the T. thermophilus-expressed recombinant cytoplasmic chromate reductase proved to be more stable under extreme chemical and thermal conditions.

[发布机构] University of the Free State