The importance of bifunctional enzymes for U(VI) reduction in Thermus scotoductus SA-01

[摘要] English: A thermophilic bacterium was isolated in 1999 by Kieft and co-workers fromgroundwater sampled at a depth of 3.2 kmbls in Mponeng, Republic of SouthAfrica, which was later identified asThermus scotoducts SA-01. T. scotoductusSA-01 has shown the ability to reduce certain metals under growth and nongrowthconditions, including Mn(IV), Co(III)-EDTA, Cr(VI)and even uranium(VI).T. scotoductus SA-01 was grown in the presence of up to 1.25 mM uranium(VI),the specific growth rate decreased with the increasing uranium concentrations. Adramatic decline is seen at 0.5 mM, when compared to the control, where anapparent lag phase was observed. TEM and EDS analyses, subsequent togrowth of T. scotoductus in uranium-containing medium, clearly show uraniummetal clusters associated extracellularly with the cells.Uranium reduction assays with the Br-PADAP complexing agent, showed that T.scotoductus SA-01 has the ability to reduce 0.25 mM of uranium in under 20hours. The whole cell reduction under non-growth conditions exhibited anoptimum temperature of 65-70 °C and an optimum pH of 7-8. Unfortunatly 2Delectrophoresis done on cultures grown in the absence and presence ofuranium(VI) showed no dramatic shifts in protein expression. This, as well as thefact that uranium has no metabolic function, led us to believe that the proteininvolved in uranium reduction was more than likely a protein with anotherfunction but which can also reduce uranium and will not be expressed due tostressful conditions such as the presence of uranium.Screening for uranium(VI) reduction activity in the subcellulare fractions of cells,namely the periplasmic, cytoplasmic and membrane fractions, led to thediscovery that activity is present in the periplasmic and membrane fractions. Forfurther separation of these fractions chromatographic methods were applied andthrough a combination of anion and cation exchange columns a protein wasidentified which might be responsible for the uranium reduction activity. Thisprotein was sent for MS/MS and N-terminal sequence determination and both ofthese methods identified the protein in question as a peptide ABC transporter,peptide binding protein. This was quite an interesting find since all work done inliterature has pointed to cytochromes c-type proteins as the 'uranium reductasesbut work done in our lab has previously shown this protein to be capable ofreducing gold, thus we know it can function as a 'reductase.The protein in question was expressed in Escherichia coli and purified usingnickel affinity chromatography and uranium(VI) reduction activity wasdetermined. Through structure modelling a disulphide bond believed to beresponsible for uranium(VI) reduction was identified. Before uranium(VI)reduction was monitored over time, the disulfide moiety of the protein wasreduced using β-mercaptoethanol. Just like the paramaters observed for wholecell reduction, the protein reduced uranium(VI) at an optimum of 65-70 °C and anoptimum pH of 7-8.The aim of this study, namely to identify a protein involved in uranium(VI)reduction from Thermus scotoductus SA-01 was successfully completed.

[发布机构] University of the Free State