Tadayuki Imanaka

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 degrees C (with a half-life of approximately 30 min at 80 degrees C), a 75% decrease in activity was observed after 2.5 min at 85 degrees 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 degrees 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 degrees C) and could not be detected at 80 to 90 degrees 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.

The hyperthermophilic archaeon Thermococcus kodakaraensis harbors a type III ribulose 1,5-bisphosphate carboxylase/oxygenase (Rbc _Tk ). It has previously been shown that Rbc _Tk is capable of supporting photoautotrophic and photoheterotrophic growth in a mesophilic host cell, Rhodopseudomonas palustris Δ3, whose three native Rubisco genes had been disrupted. Here, we have examined the enzymatic properties of Rbc _Tk at 25°C and have constructed mutant proteins in order to enhance its performance in mesophilic host cells. Initial sites for mutagenesis were selected by focusing on sequence differences in the loop 6 and α-helix 6 regions among Rbc _Tk and the enzymes from spinach (mutant proteins SP1 to SP7), Galdieria partita (GP1 and GP2), and Rhodospirillum rubrum (RR1). Loop 6 of Rbc _Tk is one residue longer than those found in the spinach and G. partita enzymes, and replacing Rbc _Tk loop 6 with these regions led to dramatic decreases in activity. Six mutant enzymes retaining significant levels of Rubisco activity were selected, and their genes were introduced into R. palustris Δ3. Cells harboring mutant protein SP6 displayed a 31% increase in the specific growth rate under photoheterotrophic conditions compared to cells harboring wild-type Rbc _Tk . SP6 corresponds to a complete substitution of the original α-helix 6 of Rbc _Tk with that of the spinach enzyme. Compared to wild-type Rbc _Tk , the purified SP6 mutant protein exhibited a 30% increase in turnover number ( k _cat ) of the carboxylase activity and a 17% increase in the k _cat / K _m value. Based on these results, seven further mutant proteins were designed and examined. The results confirmed the importance of the length of loop 6 in Rbc _Tk and also led to the identification of specific residue changes that resulted in an increase in the turnover number of Rbc _Tk at ambient temperatures.

We have developed a gene disruption system in the hyperthermophilic archaeonThermococcus kodakaraensisusing the antibiotic simvastatin and a fusion gene designed to overexpress the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene (hmg_Tk) with the glutamate dehydrogenase promoter. With this system, we disrupted theT. kodakaraensisamylopullulanase gene (apu_Tk) or a gene cluster which includesapu_Tkand genes encoding components of a putative sugar transporter. Disruption plasmids were introduced into wild-typeT. kodakaraensisKOD1 cells, and transformants exhibiting resistance to 4 μM simvastatin were isolated. The transformants exhibited growth in the presence of 20 μM simvastatin, and we observed a 30-fold increase in intracellular HMG-CoA reductase activity. The expected gene disruption via double-crossover recombination occurred at the target locus, but we also observed recombination events at thehmg_Tklocus when the endogenoushmg_Tkgene was used. This could be avoided by using the corresponding gene fromPyrococcus furiosus(hmg_Pf) or by linearizing the plasmid prior to transformation. While both gene disruption strains displayed normal growth on amino acids or pyruvate, cells without the sugar transporter genes could not grow on maltooligosaccharides or polysaccharides, indicating that the gene cluster encodes the only sugar transporter involved in the uptake of these compounds. The Δapu_Tkstrain could not grow on pullulan and displayed only low levels of growth on amylose, suggesting that Apu_Tkis a major polysaccharide-degrading enzyme inT. kodakaraensis.

We report in this study an improved system for cell-free protein synthesis at high temperatures using the lysate of Thermococcus kodakaraensis. Previous work indicated that cell-free protein synthesis of ChiAΔ4, a derivative of T. kodakaraensis chitinase, was observed within a temperature range of 40–80°C, and the maximum yield of the ChiAΔ4 synthesized was approximately 1.3 μg/ml. To increase productivity of the system, the following approaches were taken. First, the process of lysate preparation was examined, and we found that omitting the preincubation (runoff) step was especially effective to increase the translational activity of lysate. Second, the concentrations of each reaction mixture were optimized. Among them, the requirement of a high concentration of potassium acetate (250 mM) was characteristic to the T. kodakaraensis system. Third, a mutant strain of T. kodakaraensis was constructed in which a heat shock transcriptional regulator gene, phr, was disrupted. By using the lysate made from the mutant, we observed an increase in the optimum reaction temperature by 5°C. Through these modifications to the system, the yield of ChiAΔ4 was dramatically increased to 115.4 μg/ml in a batch reaction at 65°C, which was about 90 times higher than that in the previous study. Moreover, in the optimized system, a high speed of protein synthesis was achieved: over 100 μg/ml of ChiAΔ4 was produced in the first 15 min of reaction. These results indicate that the system for cell-free protein synthesis based on T. kodakaraensis lysate has a high production potential comparable to the Escherichia coli system.

Branching enzyme (BE) catalyzes formation of the branch points in glycogen and amylopectin by cleavage of the α-1,4 linkage and its subsequent transfer to the α-1,6 position. We have identified a novel BE encoded by an uncharacterized open reading frame (TK1436) of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. TK1436 encodes a conserved protein showing similarity to members of glycoside hydrolase family 57 (GH-57 family). At the C terminus of the TK1436 protein, two copies of a helix-hairpin-helix (HhH) motif were found. TK1436 orthologs are distributed in archaea of the order Thermococcales , cyanobacteria, some actinobacteria, and a few other bacterial species. When recombinant TK1436 protein was incubated with amylose used as the substrate, a product peak was detected by high-performance anion-exchange chromatography, eluting more slowly than the substrate. Isoamylase treatment of the reaction mixture significantly increased the level of short-chain α-glucans, indicating that the reaction product contained many α-1,6 branching points. The TK1436 protein showed an optimal pH of 7.0, an optimal temperature of 70°C, and thermostability up to 90°C, as determined by the iodine-staining assay. These properties were the same when a protein devoid of HhH motifs (the TK1436ΔH protein) was used. The average molecular weight of branched glucan after reaction with the TK1436ΔH protein was over 100 times larger than that of the starting substrate. These results clearly indicate that TK1436 encodes a structurally novel BE belonging to the GH-57 family. Identification of an overlooked BE species provides new insights into glycogen biosynthesis in microorganisms.

We have isolated a poly(vinyl alcohol) (PVA)-degrading bacterium from an activated sludge sample obtained from the drainage of a dyeing factory. Enrichment cultures were performed in media containing PVA as the sole or major carbon source. After several rounds of cultivation on liquid and solid media, we were able to isolate a single colony with PVA-degrading ability (strain PVA3). The bacterium could degrade PVA in the absence of symbionts or cofactors such as pyrroloquinoline quinone (PQQ). Over 90% of PVA, at an initial concentration of 0.1%, was degraded within a 6-day cultivation. Degradation was confirmed by both iodometric methods and gel permeation chromatography. Examination of the PVA attached to the cells revealed a large increase in carbonyl groups, suggesting the oxidation of hydroxyl groups of the polymer on the surfaces of cells. Addition of PQQ to the culture medium did not enhance the growth and the PVA-degrading rates of strain PVA3. Furthermore, we found that cells grown on PVA generated hydrogen peroxide upon the addition of PVA. The results strongly suggest that the initial oxidation of PVA is mediated via a PVA oxidase, and not a PQQ-dependent dehydrogenase. A biochemical and phylogenetic characterization of the bacterium was performed. The sequence of the 16S ribosomal RNA gene of the bacterium indicated a phylogenetic position of the strain within the genus Sphingopyxis, and the strain was therefore designated Sphingopyxis sp. PVA3.