Methionine Sulfoxide Reductase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis , an Enzyme Designed To Function at Suboptimal Growth Temperatures

Abstract

Methionine sulfoxide reductase (Msr) catalyzes the thioredoxin-dependent reduction and repair of methionine sulfoxide (MetO). Although Msr genes are not present in most hyperthermophile genomes, an Msr homolog encoding an MsrA-MsrB fusion protein (MsrAB Tk ) was present on the genome of the hyperthermophilic archaeon Thermococcus kodakaraensis. Recombinant proteins corresponding to MsrAB Tk and the individual domains (MsrA Tk and MsrB Tk ) were produced, purified, and biochemically examined. MsrA Tk and MsrB Tk displayed strict substrate selectivity for Met-S-O and Met-R-O, respectively. MsrAB Tk , and in particular the MsrB domain of this protein, displayed an intriguing behavior for an enzyme from a hyperthermophile. While MsrAB Tk was relatively stable at temperatures up to 80°C (with a half-life of ϳ30 min at 80°C), a 75% decrease in activity was observed after 2.5 min at 85°C, the optimal growth temperature of this archaeon. Moreover, maximal levels of MsrB activity of MsrAB Tk were observed at the strikingly low temperature of 30°C, which also was observed for MsrB Tk . Consistent with the low-temperature-specific biochemical properties of MsrAB Tk , the presence of the protein was greater in T. kodakaraensis cells grown at suboptimal temperatures (60 to 70°C) and could not be detected at 80 to 90°C. We found that the amount of intracellular MsrAB Tk protein increased with exposure to higher dissolved oxygen levels, but only at suboptimal growth temperatures. While measuring background rates of the Msr enzyme reactions, we observed significant levels of MetO reduction at high temperatures without enzyme. The occurrence of nonenzymatic MetO reduction at high temperatures may explain the specific absence of Msr homologs in most hyperthermophiles. Together with the fact that the presence of Msr in T. kodakaraensis is exceptional among the hyperthermophiles, the enzyme may represent a novel strategy for this organism to deal with low-temperature environments in which the dissolved oxygen concentrations increase.Reactive oxygen species are harmful to the cell by oxidizing various cell components, such as lipids, nucleic acids, and proteins. Among the amino acids, methionine (Met) residues are known to be particularly susceptible to oxidative stress and are easily oxidized to methionine sulfoxides (MetO). Methionine sulfoxide reductase (Msr) is an enzyme that repairs the oxidized methionine and catalyzes the thioredoxin-dependent reduction of MetO to Met (6). The oxidation of Met results in the formation of two asymmetric molecules, Met-S-O and Met-R-O. Each MetO is reduced by a specific and structurally distinct enzyme: Met-S-O is reduced by MsrA, and Met-R-O is reduced by MsrB. Msr proteins are considered to play vital roles in maintaining the intracellular redox balance and in the repair of oxidized proteins (5, 9). In mammals, defects in the function of Msr have been reported to result in neurological disorders and, in some cases, a decrease in life span (30,31).Although structurally distinct, MsrA and MsrB ...

Characterization of a Dihydrolipoyl Dehydrogenase Having Diaphorase Activity ofClostridium kluyveri

“…No diaphorase activity was detected when NADH was replaced by NADPH, as observed in the DLD of halophilic archaebacteria 20) and the hyperthermophilic archaeon Thermococcus kodakaraensis. 23) At high concentrations of DCPIP, the reaction mixture was too dark in color to detect a change in absorbance, leading to the limited assay conditions used in this study, as described under ''Materials and Methods.'' rBfmBC showed strong diaphorase activity with NBT as an electron acceptor, although quantitative analysis was not carried out because of insoluble precipitate (formazan) formation due to the enzyme reaction.…”

“…23) When the intrinsic fluorescence spectrum of as-isolated rBfmBC was recorded, emission peaks were observed around 330 and 510 nm (Fig. 3B).…”

Unveiling microbial life in the new deep‐sea hypersaline Lake Thetis. Part II: a metagenomic study

“…Moreover, seven distinct methionine sulphoxide reductases were identified in the interface metagenome. This enzyme catalyses the thioredoxin‐dependent reduction and repair of methionine sulphoxide (MetO) produced by damaging methionine oxidation (Fukushima et al ., 2007).…”

Structure–function relationship in an archaebacterial methionine sulphoxide reductase B

“…The latter situation would be expected at temperatures below the optimal thriving condition (65°C), where solubility of oxygen in the media is increased. Incidentally, for the hyperthermophilic archeon Thermococcus kodakaraensis a significantly increased expression of an MSRAB enzyme was reported at reduced growth temperatures (Fukushima et al ., 2007). Hence, we dare to speculate that in such organisms efficient recycling of MSRs by a specific system might not even be essential under optimal growth conditions and that it might suffice to rely upon the pool of free cysteine or even to (re)generate active enzyme via protein turnover/expression during critical phases rather than through recycling by efficient enzymatic activities.…”