Motomu Nishioka

Production of poly-β-hydroxybutyrate by thermophilic cyanobacterium, Synechococcus sp. MA19, under phosphate-limited conditions

Synechococcus sp. MA19, grown autotrophically under phosphate-limited conditions at 50 °C, produced poly-β-hydroxybutyrate (PHB) when intracellular phosphate content was 0.043–0.076mmol per g of cellular components. In the culture for 260h using Ca3(PO4)2 as a phosphate source, strain MA19 accumulated PHB at 55% (w/w) of the dry cells and the amount of PHB produced was 2.4gl−1 which was almost twice that without Ca3(PO4)2 addition.

The O6-methylguanine-DNA methyltransferase from the hyperthermophilic archaeon Pyrococcus sp. KOD1: a thermostable repair enzyme

Abstract The enzyme O6-methylguanine-DNA methyltransferase (MGMT) is the most common form of cellular defense against the biological effects of O6-methylguanine (O6-MeG) in DNA. Based on PCR amplification using primers derived from conserved amino acid sequences of MGMTs from 11 species, we isolated the DNA region coding for MGMT from the hyperthermophilic archaeon Pyrococcus sp. KOD1. The MGMT gene from KOD1 (mgtk) comprises 522 nucleotides, encoding 174 amino acid residues; its product shows considerable similarity to the corresponding mammalian, yeast and bacterial enzymes, especially around putative methyl acceptor sites. Phylogenetic analysis of MGMTs showed that archaeal MGMTs were grouped with their bacterial counterparts. The location of the MGMT gene on the KOD1 chromosome was also determined. The cloned KOD1 MGMT gene was overexpressed using the T7 RNA polymerase expression system, and the recombinant protein was purified by ammonium sulfate fractionation, heat treatment, ion-exchange chromatography and gel filtration chromatography. The purified recombinant protein was assayed for its enzyme activity by monitoring transfer of [3H]methyl groups from the substrate DNA to the MGMT protein; the activity was found to be stable at 90° C for at least 30 min. When the mgtk gene was placed under the control of the lac promoter and expressed in the methyltransferase-deficient Escherichia coli strain KT233 (Δada, Δogt) cells, a MGMT was produced. The enzyme was functional in vivo and complemented the mutant phenotype, making the cells resistant to the cytotoxic properties of the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine.

Crystal structure of intein homing endonuclease II encoded in DNA polymerase gene from hyperthermophilic archaeon Thermococcus kodakaraensis strain KOD1.

Proteinsplicingisa posttranslational process involving precise excision of anintervening protein domain, termed an intein. An inteinoften exhibits site-specific endonuclease activity, whichrecognizes and cleaves the DNA sequence lacking itscoding DNA sequence. The recognition sequences areusually asymmetrical and 12- to 40-bp long.

The gene yggE functions in restoring physiological defects of Escherichia coli cultivated under oxidative stress conditions.

ABSTRACT DNA microarray analysis showed that yfiD, yggB, and yggE genes were up-regulated when superoxide dismutase (SOD)-deficient Escherichia coli IM303 (I4) was cultivated under the oxidative stress generated by photoexcited TiO2, and pYFD, pYGB, and pYGE were constructed by inserting the respective genes into a pUC 19 vector. The content of reactive oxygen species (ROS) in IM303 (I4) cells carrying pYGE was reduced to 31% of ROS content in the control cells with pUC 19. In the culture of wild-type strain, E. coli MM294, in the medium with paraquat (10 μmol/l), maximum specific growth rate of the cells with pYGE was about five times higher than that of the control cells, with a decreased ROS content in the former cells. The introduction of pYGE also suppressed the occurrence of the cells with altered amino acid requirement in the culture of MM294 cells with paraquat.

Investigating a Catalytic Mechanism of Hyperthermophilic L-Threonine Dehydrogenase from Pyrococcus horikoshii

Based on our first structural data of L-threonine dehydrogenase (TDH) of Pyrococcus horikoshii (PhTDH), we examined its catalytic mechanism. The structural analysis indicated that a catalytic zinc atom at the active centre of PhTDH is coordinated by four residues (Cys42, His67, Glu68 and Glu152) with low affinity. These residues are highly conserved in alcohol dehydrogenases (ADHs) and TDHs. Several PhTDH mutants were prepared with respect to Glu152 and other residues, relating to the proton relay system that is substantially a rate-limiting step in ADH. It was found that the E152D mutant showed 3-fold higher turnover rate and reduced affinities toward L-threonine and NAD(+), compared to wild-type PhTDH. The kinetic analysis of Glu152 mutants indicated that the carboxyl group of Glu152 is important for expressing the catalytic activity. The results obtained from pH dependency of kinetic parameters suggested that Glu152 to Asp substitution causes the enhancement of deprotonation of His47 or ionization of zinc-bound water and threonine in the enzyme-NAD(+) complex. Furthermore, it was predicted that the access of threonine substrate to the enzyme-NAD(+) complex induces a large conformational change in the active domain of PhTDH. From these results, we propose here that the proton relay system works as a catalytic mechanism of PhTDH.

Metabolic alternations in SOD-deficient Escherichia coli cells when cultivated under oxidative stress from photoexcited titanium dioxide

Superoxide dismutase (SOD)-deficient Escherichia coli was cultivated under the oxidative stress generated by photoexcited titanium dioxide. These cells showed higher growth rate and glucose consumption rate with accelerated accumulation of acetic acid in the medium, compared to the cells cultivated under the normal condition without the stress. Under the stress condition, the activity of acetate kinase and mRNA expressions of the enzymes for acetic acid production (pta and ackA) were approximately doubled, while the activity of citrate synthase and mRNA expressions of the enzymes in TCA cycle (gltA, acnA, icd, sucA, sucC, sdhA, fumA and mdh) were repressed by about half, as compared with those under the normal condition. These results suggest that the stress-suffering cells switch the metabolic pathway into a “suppressed aerobiosis”, possibly for lowering the generation of reactive oxygen species.

Characterization of thermostable aminoacylase from hyperthermophilic archaeon Pyrococcus horikoshii

The gene encoding putative aminoacylase (ORF: PH0722) in the genome sequence of a hyperthermophilic archaeon, Pyrococcus horikoshii, was cloned and overexpressed in Escherichia coli. The recombinant enzyme was determined to be thermostable aminoacylase (PhoACY), forming a homotetramer. Purified PhoACY showed the ability to release amino acid molecules from the substrates N‐acetyl‐l‐Met, N‐acetyl‐l‐Gln and N‐acetyl‐l‐Leu, but had a lower hydrolytic activity towards N‐acetyl‐l‐Phe. The kinetic parameters Km and kcat were determined to be 24.6 mm and 370 s−1, respectively, for N‐acetyl‐l‐Met at 90 °C. Purified PhoACY contained one zinc atom per subunit. EDTA treatment resulted in the loss of PhoACY activity. Enzyme activity was fully recovered by the addition of divalent metal ions (Zn2+, Mn2+ and Ni2+), and Mn2+ addition caused an alteration in substrate specificity. Site‐directed mutagenesis analysis and structural modeling of PhoACY, based on Arabidopsis thaliana indole‐3‐acetic acid amino acid hydrolase as a template, revealed that, amongst the amino acid residues conserved in PhoACY, His106, Glu139, Glu140 and His164 were related to the metal‐binding sites critical for the expression of enzyme activity. Other residues, His198 and Arg260, were also found to be involved in the catalytic reaction, suggesting that PhoACY obeys a similar reaction mechanism to that proposed for mammalian aminoacylases.

Crystallization and preliminary X-ray crystallographic analysis of archaeal O6-methylguanine-DNA methyltransferase.

Crystals of archaeal O6-methylguanine-DNA methyltransferase (MGMT) from hyperthermophilic archaeon Pyrococcus kodakaraensis strain KOD1 have been grown at room temperature using polyethylene glycol as a precipitant. The diffraction pattern of the crystal extends to 2.0 A resolution at room temperature upon exposure to Cu Kalpha radiation. The crystal belongs to the space group P212121 with unit-cell dimensions of a = 52.8, b = 86.6 and c = 39.9 A. The presence of one molecule per asymmetric unit gives a crystal volume per protein mass (Vm) of 2.3 A3 Da-1 and a solvent content of 48% by volume. A full set of X-ray diffraction data was collected to 2.0 A Bragg spacings from the native crystal.

Metabolic flux analysis of a poly-β-hydroxybutyrate producing cyanobacterium,Synechococcus sp. MA19, grown under photoautotrophic conditions

To understand the utilization property of light energy,Synechococcus sp. MA19, a poly-β-hydroxybutyrate (PHB) producer, was cultivated at the different incident light intensities of 15.3, 50.0 and 78.2 W/m2 using media with and without phosphate. From the results of metabolic flux analysis, it was found that the cell yield based on ATP synthesis was estimated as 3.5×10−3 kg-biomass/mol-ATP in these cultures. Under the examined conditions, there were no significant differences in the efficiency of light energy conversion to chemical energies estimated as ATP synthesis and reducing potential (NADH+NADPH) formation whether the PHB synthesis took place or not. The energy converted from light to ATP was kept relatively high around the energy absorbed by the cells of 2.5–3.0×106 J h−1 kg−1, whereas the energy of reducing potential was hardly changed in the examined range of the energy absorbed by the cells.

Crystallographic study of intein homing endonuclease II encoded in the archaeal DNA polymerase gene.

Intein homing endonucleases are proteins spliced out from a precursor protein and site-specific enzymes that make double-strand breaks in inteinless alleles. Crystals of intein homing endonuclease II from the hyperthermophilic archaeon Pyrococcus kodakaraensis strain KOD1 (PI-PkoII) have been grown at room temperature using ammonium sulfate as a precipitant. The diffraction pattern of the crystal extends to 3.0 A resolution at room temperature upon exposure to synchrotron X-rays at KEK-PF, Japan. The crystals have symmetry consistent with space group C222(1), with unit-cell parameters a = 107.6, b = 150.5, c = 146.8 A. A full set of X-ray diffraction data were collected to 3.0 A Bragg spacing from a native crystal with an overall R(merge) of 4.8% and a completeness of 96.6%.