Crystal structure of a glycoside hydrolase family 68 ß-fructosyltransferase from Beijerinckia indica subsp. indica in complex with fructose.

An enzyme belonging to glycoside hydrolase family 68 (GH68) from Beijerinckia indica subsp. indica NBRC 3744 was expressed in Escherichia coli. Biochemical characterization showed that the enzyme was identified to be a ß-fructosyltransferase (BiBftA). Crystallization of a full-length BiBftA was initially attempted, but no crystals were obtained. We constructed a variant in which 5 residues (Pro199-Gly203) and 13 residues (Leu522-Gln534) in potentially flexible regions were deleted, and we successfully crystallized this variant BiBftA. BiBftA is composed of a five-bladed ß-propeller fold as in other GH68 enzymes. The structure of BiBftA in complex with fructose unexpectedly indicated that one ß-fructofuranose (ß-Fruf) molecule and one ß-fructopyranose molecule bind to the catalytic pocket. The orientation of ß-Fruf at subsite -1 is tilted from the orientation observed in most GH68 enzymes, presenting a second structure of a GH68 enzyme in complex with the tilted binding mode of ß-Fruf.

A novel non-segmented double-stranded RNA virus from an Arctic isolate of Pythium polare.

We investigated virus infection in the oomycete Pythium polare from the Arctic. From 39 isolates investigated, 14 contained virus-like double-stranded RNA (dsRNA). Next generation sequencing revealed that the P. polare isolate OPU1176 contained three different virus-like sequences. We determined the full-length genome sequence of one of them. The 5397 nt-length genome had two overlapped open reading frames (ORFs) consistent with a toti and toti-like viruses, that we named Pythium polare RNA virus 1 (PpRV1). The ORF2 encoded an RNA-dependent RNA polymerase (RdRp). The shifty heptamer motif and RNA pseudoknot were predicted near the stop codon of ORF1, implying that the RdRp could be translated as a fusion protein with the ORF1 protein. Phylogenetic analysis with deduced RdRp amino acid sequences indicated that oomycete virus PpRV1 was closely related to the unclassified arthropod toti-like viruses. The comparison of PpRV1-free and -infected lines suggested that PpRV1 infected in a symptomless manner.

Rapid evaluation of 1-kestose producing ß-fructofuranosidases from Aspergillus species and enhancement of 1-kestose production using a PgsA surface-display system.

1-Kestose is a key prebiotic fructooligosaccharide (FOS) sugar. Some ß-fructofuranosidases (FFases) have high transfructosylation activity, which is useful for manufacturing FOS. Therefore, obtaining FFases that produce 1-kestose efficiently is important. Here, we established a rapid FFase evaluation method using Escherichia coli that display different FFases fused to a PgsA anchor protein from Bacillus subtilis. E. coli cell suspensions expressing the PgsA-FFase fusion efficiently produce FOS from sucrose. Using this screening technique, we found that the E. coli transformant expressing Aspergillus kawachii FFase (AkFFase) produced a larger amount of 1-kestose than those expressing FFases from A. oryzae and A. terreus. Saturation mutagenesis of AkFFase was performed, and the mutant G85W was obtained. The E. coli transformant expressing AkFFase G85W markedly increased production of 1-kestose. Our results indicate that the surface display technique using PgsA is useful for screening of FFases, and AkFFase G85W is likely to be suitable for 1-kestose production. ABBREVIATIONS: AkFFase: Aspergillus kawachii FFase; AoFFase: Aspergillus oryzae FFase; AtFFase: Aspergillus terreus FFase; FFase: ß-fructofuranosidase; FOS: fructooligosaccharide; fructosylnystose: 1F-ß-fructofuranosylnystose.

Hard x-ray telescopes to be onboard ASTRO-H.

The new Japanese x-ray astronomy satellite, ASTRO-H, will carry two identical hard x-ray telescopes (HXTs), which cover the energy range of 5 to 80 keV. The HXT mirrors employ tightly nested, conically approximated thin-foil Wolter-I optics, and the mirror surfaces are coated with Pt/C depth-graded multilayers to enhance the hard x-ray effective area by means of Bragg reflection. The HXT comprises foils 120-450 mm in diameter and 200 mm in length, with a focal length of 12 m. To obtain a large effective area, 213 aluminum foils 0.2 mm in thickness are tightly nested confocally. The requirements for HXT are a total effective area of >300 cm2 at 30 keV and an angular resolution of <1.7' in half-power diameter (HPD). Fabrication of two HXTs has been completed, and the x-ray performance of each HXT was measured at a synchrotron radiation facility, SPring-8 BL20B2 in Japan. Angular resolutions (HPD) of 1.9' and 1.8' at 30 keV were obtained for the full telescopes of HXT-1 and HXT-2, respectively. The total effective area of the two HXTs at 30 keV is 349 cm2.

Design and fabrication of a supermirror with smooth and broad response for hard x-ray telescopes.

We present our design and fabrication scheme of a supermirror for hard x-ray telescopes to improve the performance of reflection. Supermirrors are designed to achieve target reflectivity profiles with small ripples in the interesting energy range with a limited number of layer pairs. Starting from conventional design methods, we optimized the thickness distribution of the structure using a numerical approach with the merit function established for this application. As an example, a platinum-carbon supermirror structure of 123 layer pairs with a varying d-space of 2-6 nm was designed to enhance the reflectivity in the energy range from 11 to 55 keV with a flat top at a grazing angle of 0.28 deg. The multilayer thus designed was deposited on a float glass by our DC magnetron sputtering system. The reflectivity profile was successfully measured to be ~20% with little oscillation in all energy band of interest.

Purification and characterization of arylmalonate decarboxylase from Achromobacter sp. KU1311.

We have isolated, purified and characterized arylmalonate decarboxylase (AMDase; EC 4.1.1.76). This is an unique enzyme that gives optically pure arylpropionates from the corresponding arylmalonates. Recently, we have screened similar enzyme producers from soil samples and succeeded in isolating Achromobacter sp. KU1311. The gene encoding the enzyme was cloned and sequenced. The AMDase gene consists of 720 nucleotides, which specifies a 240 amino acid protein with a relative molecular mass of 24,735. This enzyme was purified and its characteristics were compared with those of the hitherto known enzyme from Alcaligenes bronchisepticus KU1201.

Production and performance of the inFOCmicroS 20-40-keV graded multilayer mirror.

The International Focusing Optics Collaboration for microCrab Sensitivity (InFOCmicroS) balloonborne hard x-ray telescope incorporates graded Pt/C multilayers replicated onto segmented Al foils to obtain the significant effective area at energies previously inaccessible to x-ray optics. Reflectivity measurements of individual foils demonstrate our capability to produce a mass quantity of multilayered foils with a rms roughness of 0.5 nm. The effective area of the completed mirror is 78 and 22 cm2 at 20 and 40 keV, respectively. The measured half-power diameter is 2.0 +/- 0.6 are min (90% confidence). The successful completion of this mirror demonstrates its applicability to future x-ray telescopes such as Constellation-X.

Characterization of the supermirror hard-x-ray telescope for the InFOCmuS balloon experiment.

A hard-x-ray telescope is successfully produced for balloon observations by making use of depth-graded multilayers, or so-called supermirrors, with platinum-carbon (Pt/C) layer pairs. It consists of four quadrant units assembled in an optical configuration with a diameter of 40 cm and a focal length of 8 m. Each quadrant is made of 510 pieces of coaxially and confocally aligned supermirrors that significantly enhance the sensitivity in an energy range of 20-40 keV. The configuration of the telescope is similar to the x-ray telescope onboard Astro-E, but with a longer focal length. The reflectivity of supermirrors is of the order of 40% in the energy range concerned at a grazing angle of 0.2 deg. The effective area of a fully assembled telescope is 50 cm2 at 30 keV. The angular resolution is 2.37 arc min at half-power diameter 8.0 keV. The field of view is 12.6 arc min in the hard-x-ray region, depending somewhat on x-ray energies. We discuss these characteristics, taking into account the figure errors of reflectors and their optical alignment in the telescope assembly. This hard-x-ray telescope is unanimously afforded in the International Focusing Optics Collaboration for muCrab Sensitivity balloon experiment.