Biogeochemical transformation of mercury driven by microbes involved in anaerobic digestion of municipal wastewater.

Anaerobic digestion (AD) with municipal wastewater contained heavy metal mercury (Hg) highly affects the utilization of activated sludge, and poses severe threat to the health of human beings. However, the biogeochemical transformation of Hg during AD remains unclear. Here, we investigated the biogeochemical transformation and environmental characteristics of Hg and the variations of dominant microbes during AD. The results showed that Hg(II) methylation is dominant in the early stage of AD, while methylmercury (MeHg) demethylation dominates in the later stage. Dissolved total Hg (DTHg) in the effluent sludge decreased with time, while THg levels enhanced to varying degrees at the final stage. Sulfate significant inhibits MeHg formation, reduces bioavailability of Hg(II) by microbes and thus inhibits Hg(II) methylation. Microbial community analysis reveals that strains in Methanosarcina and Aminobacterium from the class of Methanomicrobia, rather than Deltaproteobacteria, may be directly related to Hg(II) methylation and MeHg demethylation. Overall, this research provide insights into the biogeochemical transformation of Hg in the anaerobic digestion of municipal wastewater treatment. This work is beneficial for scientific treatment of municipal wastewater and effluent sludge, thus reducing the risk of MeHg to human beings.

Random mutagenesis of a hyperthermophilic archaeon identified tRNA modifications associated with cellular hyperthermotolerance.

Random mutagenesis for the hyperthermophilic archaeon Thermococcus kodakarensis was established by the insertion of an artificial transposon designed to allow easy identification of the transposon-inserted locus. The phenotypic screening was applied for the isolation of thermosensitive mutants of T. kodakarensis, which resulted in the isolation of 16 mutants showing defective growth at the supraoptimal temperature 93°C. The high occurrence of the mutants suggested that the high thermotolerance of hyperthermophiles was achieved by a combination of diverse gene functions. The transposon insertion sites in two-thirds of the mutants were identified in a group of genes responsible for tRNA modifications including 7-formamidino-7-deaza-guanosine (archaeosine), N1-methyladenosine/N1-methylinosine, N4-acetylcytidine, and N2-dimethylguanosine/N2,N2-dimethylguanosine. LC-MS/MS analyses of tRNA nucleosides and fragments exhibited disappearance of the corresponding modifications in the mutants. The melting temperature of total tRNA fraction isolated from the mutants lacking archaeosine or N1-methyladenosine/N1-methylinosine decreased significantly, suggesting that the thermosensitive phenotype of these mutants was attributed to low stability of the hypomodified tRNAs. Genes for metabolism, transporters, and hypothetical proteins were also identified in the thermosensitive mutants. The present results demonstrated the usefulness of random mutagenesis for the studies on the hyperthermophile, as well as crucial roles of tRNA modifications in cellular thermotolerance.

Crystal structure of pantoate kinase from Thermococcus kodakarensis.

The coenzyme A biosynthesis pathways in most archaea involve two unique enzymes, pantoate kinase and phosphopantothenate synthetase, to convert pantoate to 4'-phosphopantothenate. Here, we report the first crystal structure of pantoate kinase from the hyperthermophilic archaeon, Thermococcus kodakarensis and its complex with ATP and a magnesium ion. The electron density for the adenosine moiety of ATP was very weak, which most likely relates to its broad nucleotide specificity. Based on the structure of the active site that contains a glycerol molecule, the pantoate binding site and the roles of the highly conserved residues are suggested.

The metal transporter CrNRAMP1 is involved in zinc and cobalt transports in Chlamydomonas reinhardtii.

Metal homeostasis is essential cellular progress for cell growth. Metal ion transporters play important roles in the first line of defense to cellular metal homeostasis perturbations. NRAMP transporter family was one of the most important classes in plant cells. However, functions and substrate specificities of the NRAMP family remain unknown in Chlamydomonas reinhardtii, a model unicellular plant. In this study, we identified CrNRAMP1 as an important transporter involved in zinc and cobalt transport. Heterologous and homologous functional analyses of CrNRAMP1 showed that CrNRAMP1 plays important roles in zinc and cobalt homeostasis. The expression of CrNRAMP1 correlated with zinc or cobalt concentrations, but excluding cadmium. These results help to understand the functions and specificities of NRAMP family members in C. reinhardtii.

An ornithine ω-aminotransferase required for growth in the absence of exogenous proline in the archaeon Thermococcus kodakarensis.

Aminotransferases are pyridoxal 5'-phosphate-dependent enzymes that catalyze reversible transamination reactions between amino acids and α-keto acids, and are important for the cellular metabolism of nitrogen. Many bacterial and eukaryotic ω-aminotransferases that use l-ornithine (Orn), l-lysine (Lys), or γ-aminobutyrate (GABA) have been identified and characterized, but the corresponding enzymes from archaea are unknown. Here, we examined the activity and function of TK2101, a gene annotated as a GABA aminotransferase, from the hyperthermophilic archaeon Thermococcus kodakarensis We overexpressed the TK2101 gene in T. kodakarensis and purified and characterized the recombinant protein and found that it displays only low levels of GABA aminotransferase activity. Instead, we observed a relatively high ω-aminotransferase activity with l-Orn and l-Lys as amino donors. The most preferred amino acceptor was 2-oxoglutarate. To examine the physiological role of TK2101, we created a TK2101 gene-disruption strain (ΔTK2101), which was auxotrophic for proline. Growth comparison with the parent strain KU216 and the biochemical characteristics of the protein strongly suggested that TK2101 encodes an Orn aminotransferase involved in the biosynthesis of l-Pro. Phylogenetic comparisons of the TK2101 sequence with related sequences retrieved from the databases revealed the presence of several distinct protein groups, some of which having no experimentally studied member. We conclude that TK2101 is part of a novel group of Orn aminotransferases that are widely distributed at least in the genus Thermococcus, but perhaps also throughout the Archaea.

Branched-Chain Polyamine Found in Hyperthermophiles Induces Unique Temperature-Dependent Structural Changes in Genome-Size DNA.

A pentavalent branched-chain polyamine, N4 -bis(aminopropyl)spermidine 3(3)(3)4, is a unique polycation found in the hyperthermophilic archaeon Thermococcus kodakarensis, which grows at temperatures between 60 and 100 °C. We studied the effects of this branched-chain polyamine on DNA structure at different temperatures up to 80 °C. Atomic force microscopic observation revealed that 3(3)(3)4 induces a mesh-like structure on a large DNA (166 kbp) at 24 °C. With an increase in temperature, DNA molecules tend to unwind, and multiple nano-loops with a diameter of 10-50 nm are generated along the DNA strand at 80 °C. These results were compared to those obtained with linear-chain polyamines, homocaldopentamine 3334 and spermidine, the former of which is a structural isomer of 3(3)(3)4. These specific effects are expected to neatly concern with its role on high-temperature preference in hyperthermophiles.

Pcal_0632, a phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Pyrobaculum calidifontis.

Genome sequence of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_0632, annotated as glyceraldehyde-3-phosphate dehydrogenase, which is partially overlapped with phosphoglycerate kinase. In the phylogenetic tree, Pcal_0632 clustered with phosphorylating glyceraldehyde-3-phosphate dehydrogenases characterized from hyperthermophilic archaea and exhibited highest identity of 54% with glyceraldehyde-3-phosphate dehydrogenase from Sulfolobus tokodaii. To examine biochemical function of the protein, Pcal_0632 gene was expressed in Escherichia coli and the gene product was purified. The recombinant enzyme catalyzed the conversion of glyceraldehyde 3-phosphate and inorganic phosphate into 1,3-bisphosphoglycerate utilizing both NAD and NADP as cofactor with a marked preference for NADP. The enzyme was highly stable against temperature and denaturants. Half-life of the enzyme was 60 min at 100 °C. It retained more than 60% of its activity even after an incubation of 72 h at room temperature in the presence of 6 M urea. High thermostability and resistance against denaturants make Pcal_0632 a novel glyceraldehyde-3-phosphate dehydrogenase.

Structural basis of a Ni acquisition cycle for [NiFe] hydrogenase by Ni-metallochaperone HypA and its enhancer.

The Ni atom at the catalytic center of [NiFe] hydrogenases is incorporated by a Ni-metallochaperone, HypA, and a GTPase/ATPase, HypB. We report the crystal structures of the transient complex formed between HypA and ATPase-type HypB (HypBAT) with Ni ions. Transient association between HypA and HypBAT is controlled by the ATP hydrolysis cycle of HypBAT, which is accelerated by HypA. Only the ATP-bound form of HypBAT can interact with HypA and induces drastic conformational changes of HypA. Consequently, upon complex formation, a conserved His residue of HypA comes close to the N-terminal conserved motif of HypA and forms a Ni-binding site, to which a Ni ion is bound with a nearly square-planar geometry. The Ni binding site in the HypABAT complex has a nanomolar affinity (Kd = 7 nM), which is in contrast to the micromolar affinity (Kd = 4 µM) observed with the isolated HypA. The ATP hydrolysis and Ni binding cause conformational changes of HypBAT, affecting its association with HypA. These findings indicate that HypA and HypBAT constitute an ATP-dependent Ni acquisition cycle for [NiFe]-hydrogenase maturation, wherein HypBAT functions as a metallochaperone enhancer and considerably increases the Ni-binding affinity of HypA.

Engineering of the Hyperthermophilic Archaeon Thermococcus kodakarensis for Chitin-Dependent Hydrogen Production.

Thermococcus kodakarensis is a hyperthermophilic archaeon that harbors a complete set of genes for chitin degradation to fructose 6-phosphate. However, wild-type T. kodakarensis KOD1 does not display growth on chitin. In this study, we developed a T. kodakarensis strain that can grow on chitin via genetic and adaptive engineering. First, a chitinase overproduction strain (KC01) was constructed by replacing the chitinase gene promoter with a strong promoter from the cell surface glycoprotein gene, resulting in increased degradation of swollen chitin and accumulation of N-,N'-diacetylchitobiose in the medium. To enhance N-,N'-diacetylchitobiose assimilation in KC01, genes encoding diacetylchitobiose deacetylase, exo-ß-d-glucosaminidase, and glucosamine-6-phosphate deaminase were also overexpressed to obtain strain KC04. To strengthen the glycolytic flux of KC04, the gene encoding Tgr (transcriptional repressor of glycolytic genes) was disrupted to obtain strain KC04Δt. In both KC04 and KC04Δt strains, degradation of swollen chitin was further enhanced. In the culture broth of these strains, the accumulation of glucosamine was observed. KC04Δt was repeatedly inoculated in a swollen-chitin-containing medium for 13 cultures. This adaptive engineering strategy resulted in the isolation of a strain (KC04ΔtM1) that showed almost complete degradation of 0.4% (wt/vol) swollen chitin after 90 h. The strain produced high levels of acetate and ammonium in the culture medium, and, moreover, molecular hydrogen was generated. This strongly suggests that strain KC04ΔtM1 has acquired the ability to convert chitin to fructose 6-phosphate via deacetylation and deamination and further convert fructose 6-phosphate to acetate via glycolysis coupled to hydrogen generation.IMPORTANCE Chitin is a linear homopolymer of ß-1,4-linked N-acetylglucosamine and is the second most abundant biomass next to cellulose. Compared to the wealth of research focused on the microbial degradation and conversion of cellulose, studies addressing microbial chitin utilization are still limited. In this study, using the hyperthermophilic archaeon Thermococcus kodakarensis as a host, we have constructed a strain that displays chitin-dependent hydrogen generation. The apparent hydrogen yield per unit of sugar consumed was slightly higher with swollen chitin than with starch. As gene manipulation in T. kodakarensis is relatively simple, the strain constructed in this study can also be used as a parent strain for the development and expansion of chitin-dependent biorefinery, in addition to its capacity to produce hydrogen.

A pentose bisphosphate pathway for nucleoside degradation in Archaea.

Owing to the absence of the pentose phosphate pathway, the degradation pathway for the ribose moieties of nucleosides is unknown in Archaea. Here, in the archaeon Thermococcus kodakarensis, we identified a metabolic network that links the pentose moieties of nucleosides or nucleotides to central carbon metabolism. The network consists of three nucleoside phosphorylases, an ADP-dependent ribose-1-phosphate kinase and two enzymes of a previously identified NMP degradation pathway, ribose-1,5-bisphosphate isomerase and type III ribulose-1,5-bisphosphate carboxylase/oxygenase. Ribose 1,5-bisphosphate and ribulose 1,5-bisphosphate are intermediates of this pathway, which is thus designated the pentose bisphosphate pathway.

Double-Strand Breaks in Genome-Sized DNA Caused by Ultrasound.

DNA double-strand breaks (DSBs) caused by ultrasound were evaluated in a quantitative manner by single-molecule fluorescence microscopy. We compared the effect of time-interval (or pulse) sonication to that of continuous wave (CW) sonication at a fixed frequency of 30 kHz. Pulses caused fewer DSBs than CW sonication under the same total input ultrasound energy when the pulse repetition period was above the order of a second. In contrast, pulses caused more DSBs than CW sonication for pulse widths shorter than a second. These effect of ultrasound on DNA were interpreted in terms of the time-dependent decay in the probability of breakage during the duration of a pulse. We propose a simple phenomenological model by considering a characteristic decay in the probability of DSBs during single-pulse sonication, which reproduces the essence of the experimental trend. In addition, a data analysis revealed a characteristic scaling behavior between the number of pulses and the number of DSBs.

Pcal_1311, an alcohol dehydrogenase homologue from Pyrobaculum calidifontis, displays NADH-dependent high aldehyde reductase activity.

Genome sequence of Pyrobaculum calidifontis, a hyperthermophilic archaeon, harbors three open-reading frames annotated as alcohol dehydrogenases. One of them, Pcal_1311, does not display a significantly high homology with any of the characterized alcohol dehydrogenases. Highest homology of 38% was found with the characterized counterpart from Geobacillus stearothermophilus. To examine the biochemical properties of Pcal_1311, we have cloned and functionally expressed the gene in Escherichia coli. Purified recombinant Pcal_1311 catalyzed the NAD(H)-dependent oxidation of various alcohols and reduction of aldehydes, with a marked preference for substrates with functional group at the terminal carbon. Highest activity for the oxidation reaction (3 µmol min-1 mg-1) was found with 1,4-butanediol and for the reduction reaction (150 µmol min-1 mg-1) with glutaraldehyde. Both the oxidation and reduction activities increased with the increase in temperature up to 80 °C. Recombinant Pcal_1311 was highly stable and retained more than 90% activity even after incubation of 180 min at 90 °C. In addition to the thermostabilty, Pcal_1311 was highly stable in the presence of known denaturants including urea and guanidine hydrochloride. The high stability, particularly thermostability, and the NADH-dependent aldehyde reduction activity make Pcal_1311 a unique member in the alcohol dehydrogenase family.

Pcal_0111, a highly thermostable bifunctional fructose-1,6-bisphosphate aldolase/phosphatase from Pyrobaculum calidifontis.

Pyrobaculum calidifontis genome harbors an open reading frame Pcal_0111 annotated as fructose bisphosphate aldolase. Although the gene is annotated as fructose bisphosphate aldolase, it exhibits a high homology with previously reported fructose-1,6-bisphosphate aldolase/phosphatase from Thermoproteus neutrophilus. To examine the biochemical properties of Pcal_0111, we have cloned and expressed the gene in Escherichia coli. Purified recombinant Pcal_0111 catalyzed both phosphatase and aldolase reactions with specific activity values of 4 U and 1.3 U, respectively. These values are highest among the fructose 1,6-bisphosphatases/aldolases characterized from archaea. The enzyme activity increased linearly with the increase in temperature until 100 °C. Recombinant Pcal_0111 is highly stable with a half-life of 120 min at 100 °C. There was no significant change in the circular dichroism spectra of the protein up to 90 °C. The enzyme activity was not affected by AMP but strongly inhibited by ATP with an IC50 value of 0.75 mM and mildly by ADP. High thermostability and inhibition by ATP make Pcal_0111 a unique fructose 1,6-bisphosphatase/aldolase.

Gene regulation of two ferredoxin:NADP+ oxidoreductases by the redox-responsive regulator SurR in Thermococcus kodakarensis.

The redox-responsive regulator SurR in the hyperthermophilic archaea Pyrococcus furiosus and Thermococcus kodakarensis binds to the SurR-binding consensus sequence (SBS) by responding to the presence of elemental sulfur. Here we constructed a surR gene disruption strain (DTS) in T. kodakarensis, and identified the genes that were under SurR control by comparing the transcriptomes of DTS and parent strains. Among these genes, transcript levels of ferredoxin:NADP+ oxidoreductases 1 and 2 (FNOR1 and FNOR2) genes displayed opposite responses to surR deletion, indicating that SurR repressed FNOR1 transcription while enhancing FNOR2 transcription. Each promoter region contains an SBS upstream (uSBS) and downstream (dSBS) of TATA. In addition to in vitro binding assays, we examined the roles of each SBS in vivo. In FNOR1, mutations in either one of the SBSs resulted in a complete loss of repression, indicating that the presence of both SBSs was essential for repression. In FNOR2, uSBS indeed functioned to enhance gene expression, whereas dSBS functioned in gene repression. SurR bound to uSBS2 of FNOR2 more efficiently than to dSBS2 in vitro, which may explain why SurR overall enhances FNOR2 transcription. Further analyses indicated the importance in the distance between uSBS and TATA for transcriptional activation in FNOR2.

Genetic analyses of the functions of [NiFe]-hydrogenase maturation endopeptidases in the hyperthermophilic archaeon Thermococcus kodakarensis.

The maturation of [NiFe]-hydrogenases requires a number of accessory proteins, which include hydrogenase-specific endopeptidases. The endopeptidases carry out the final cleavage reaction of the C-terminal regions of [NiFe]-hydrogenase large subunit precursors. The hyperthermophilic archaeon Thermococcus kodakarensis harbors two [NiFe]-hydrogenases, a cytoplasmic Hyh and a membrane-bound Mbh, along with two putative hydrogenase-specific endopeptidase genes. In this study, we carried out a genetic examination on the two endopeptidase genes, TK2004 and TK2066. Disruption of TK2004 resulted in a strain that could not grow under conditions requiring hydrogen evolution. The Mbh large subunit precursor (pre-MbhL) in this strain was not processed at all whereas Hyh cleavage was not affected. On the other hand, disruption of TK2066 did not affect the growth of T. kodakarensis under the conditions examined. Cleavage of the Hyh large subunit precursor (pre-HyhL) was impaired, but could be observed to some extent. In a strain lacking both TK2004 and TK2066, cleavage of pre-HyhL could not be observed. Our results indicate that pre-MbhL cleavage is carried out solely by the endopeptidase encoded by TK2004. Pre-HyhL cleavage is mainly carried out by TK2066, but TK2004 can also play a minor role in this cleavage.

Identification of a novel copper-activated and halide-tolerant laccase in Geobacillus thermopakistaniensis.

Genome search of Geobacillus thermopakistaniensis, formerly Geobacillus sp. SBS-4S, revealed the presence of an open reading frame (ESU71923) annotated as laccase. However, the gene product did not display any laccase-like activity against the substrates examined. The laccase activity was, therefore, purified from G. thermopakistaniensis cells and N-terminal amino acid residues of the enzyme were determined. These residues matched the N-terminal sequence of an open reading frame annotated as a copper oxidase (ESU72270). In order to characterize the enzyme, recombinant ESU72270 was prepared in Escherichia coli. The recombinant protein was found to exhibit a negligible amount of laccase activity when produced in the absence of copper in the growth medium. However, the recombinant protein exhibited significantly high laccase activity when produced in the presence of copper. The recombinant enzyme showed highest activity at 60 °C and a pH of 7-7.5. The purified enzyme was highly tolerant to various halides and organic solvents, thus having a potential for various industrial applications. To the best of our knowledge, this is the first characterization of a laccase from genus Geobacillus which identifies a gene responsible for functional laccase in this genus.

Specific Conformational Change in Giant DNA Caused by Anticancer Tetrazolato-Bridged Dinuclear Platinum(II) Complexes: Middle-Length Alkyl Substituents Exhibit Minimum Effect.

Derivatives of the highly antitumor-active compound [{cis-Pt(NH3)2}2(µ-OH)(µ-tetrazolato-N2,N3)]2+ (5-H-Y), which is a tetrazolato-bridged dinuclear platinum(II) complex, were prepared by substituting a linear alkyl chain moiety at C5 of the tetrazolate ring. The general formula for the derivatives is [{cis-Pt(NH3)2}2(µ-OH)(µ-5-R-tetrazolato-N2,N3)]2+, where R is (CH2)nCH3 and n = 0 to 8 (complexes 1-9). The cytotoxicity of complexes 1-4 in NCI-H460 human non-small-cell lung cancer cells decreased with increasing alkyl chain length, and those of complexes 5-9 increased with increasing alkyl chain length. That is, the in vitro cytotoxicity of complexes 1-9 was found to have a U-shaped association with alkyl chain length. This U-shaped association is attributable to the degree of intracellular accumulation. Although circular dichroism spectroscopic measurement indicated that complexes 1-9 induced comparable conformational changes in the secondary structure of DNA, the tetrazolato-bridged complexes induced different degrees of DNA compaction as revealed by a single DNA measurement with fluorescence microsopy, which also had a U-shaped association with alkyl chain length that matched the association observed for cytotoxicity. Complexes 7-9, which had alkyl chains long enough to confer surfactant-like properties to the complex, induced DNA compaction 20 or 1000 times more efficiently than 5-H-Y or spermidine. A single DNA measurement with transmission electron microscopy revealed that complex 8 formed large spherical self-assembled structures that induced DNA compaction with extremely high efficiency. This result suggests that these structures may play a role in the DNA compaction that was induced by the complexes with the longer alkyl chains. The derivatization with a linear alkyl chain produced a series of complexes with unique cellular accumulation and DNA conformational change profiles and a potentially useful means of developing next-generation platinum-based anticancer drugs. In addition, the markedly high ability of these complexes to induce DNA compaction and their high intracellular accumulation emphasized the difference in mechanism of action from platinum-based anticancer drugs.

Escherichia coli Signal Peptidase Recognizes and Cleaves Archaeal Signal Sequence.

Tk1884, an open reading frame encoding α-amylase in Thermococcus kodakarensis, was cloned with the native signal sequence and expressed in Escherichia coli. Heterologous gene expression resulted in secretion of the recombinant protein to the extracellular culture medium. Extracellular α-amylase activity gradually increased after induction. Tk1884 was purified from the extracellular medium, and its molecular mass determined by electrospray ionization mass spectrometry indicated the cleavage of a few amino acids. The N-terminal amino acid sequence of the purified Tk1884 was determined, which revealed that the signal peptide was cleaved between Ala26 and Ala27 by E. coli signal peptidase. To the best of our knowledge, this is the first report describing an archaeal signal sequence recognized and cleaved by E. coli signal peptidase.

Crystal structure of a [NiFe] hydrogenase maturation protease HybD from Thermococcus kodakarensis KOD1.

A [NiFe] hydrogenase maturation protease HybD from Thermococcus kodakarensis KOD1 (TkHybD) is involved in the cleavage of the C-terminal residues of [NiFe] hydrogenase large subunits by Ni recognition. Here, we report the crystal structure of TkHybD at 1.82 Å resolution to better understand this process. TkHybD exhibits an α/ß/α sandwich fold with conserved residues responsible for the Ni recognition. Comparisons of TkHybD with homologous proteins also reveal that they share a common overall architecture, suggesting that they have similar catalytic functions. Our results including metal binding site prediction provide insight into the substrate recognition and catalysis mechanism of TkHybD. Proteins 2016; 84:1321-1327. © 2016 Wiley Periodicals, Inc.

Mutation design of a thermophilic Rubisco based on three-dimensional structure enhances its activity at ambient temperature.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a central role in carbon dioxide fixation on our planet. Rubisco from a hyperthermophilic archaeon Thermococcus kodakarensis (Tk-Rubisco) shows approximately twenty times the activity of spinach Rubisco at high temperature, but only one-eighth the activity at ambient temperature. We have tried to improve the activity of Tk-Rubisco at ambient temperature, and have successfully constructed several mutants which showed higher activities than the wild-type enzyme both in vitro and in vivo. Here, we designed new Tk-Rubisco mutants based on its three-dimensional structure and a sequence comparison of thermophilic and mesophilic plant Rubiscos. Four mutations were introduced to generate new mutants based on this strategy, and one of the four mutants, T289D, showed significantly improved activity compared to that of the wild-type enzyme. The crystal structure of the Tk-Rubisco T289D mutant suggested that the increase in activity was due to mechanisms distinct from those involved in the improvement in activity of Tk-Rubisco SP8, a mutant protein previously reported to show the highest activity at ambient temperature. Combining the mutations of T289D and SP8 successfully generated a mutant protein (SP8-T289D) with the highest activity to date both in vitro and in vivo. The improvement was particularly pronounced for the in vivo activity of SP8-T289D when introduced into the mesophilic, photosynthetic bacterium Rhodopseudomonas palustris, which resulted in a strain with nearly two-fold higher specific growth rates compared to that of a strain harboring the wild-type enzyme at ambient temperature. Proteins 2016; 84:1339-1346. © 2016 Wiley Periodicals, Inc.