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1.  Crystallization and preliminary X-ray crystallographic analysis of Pz peptidase B from Geobacillus collagenovorans MO-1 
In this work, Pz peptidase B, an intracellular M3 metallopeptidase that is found in the thermophile Geobacillus collagenovorans MO-1, was crystallized using the counter-diffusion method.
Pz peptidase B is an intracellular M3 metallopeptidase that is found together with Pz peptidase A in the thermophile Geobacillus collagenovorans MO-1 and recognizes collagen-specific tripeptide units (-Gly-Pro-X-). These peptidases have low homology in their primary structures; however, their cleavage patterns towards peptide substrates are similar. In this work, Pz peptidase B was crystallized using the counter-diffusion method. Data were collected to a resolution of 1.6 Å at 100 K from a crystal obtained in the Japanese Experiment Module (JEM; also known as ‘Kibo’) at the International Space Station (ISS). The crystal belonged to the trigonal space group P3121, with unit-cell parameters a = b = 87.64, c = 210.5 Å. A complete data set was also obtained from crystals of selenomethionine-substituted protein.
doi:10.1107/S1744309112018969
PMCID: PMC3388914  PMID: 22750857
Pz peptidase B; Geobacillus collagenovorans MO-1; microgravity
2.  Impact of Site-Directed Mutant Luciferase on Quantitative Green and Orange/Red Emission Intensities in Firefly Bioluminescence 
Scientific Reports  2013;3:2490.
Firefly bioluminescence has attracted great interest because of its high quantum yield and intriguing modifiable colours. Modifications to the structure of the enzyme luciferase can change the emission colour of firefly bioluminescence, and the mechanism of the colour change has been intensively studied by biochemists, structural biologists, optical physicists, and quantum-chemistry theorists. Here, we report on the quantitative spectra of firefly bioluminescence catalysed by wild-type and four site-directed mutant luciferases. While the mutation caused different emission spectra, the spectra differed only in the intensity of the green component (λmax ~ 560 nm). In contrast, the orange (λmax ~ 610 nm) and red (λmax ~ 650 nm) components present in all the spectra were almost unaffected by the modifications to the luciferases and changes in pH. Our results reveal that the intensity of the green component is the unique factor that is influenced by the luciferase structure and other reaction conditions.
doi:10.1038/srep02490
PMCID: PMC3750616  PMID: 23970036
3.  Crystallization and preliminary X-ray crystallographic studies of Pz peptidase A from Geobacillus collagenovorans MO-1 
Pz peptidase A has been cocrystallized with a phosphine peptide inhibitor (PPI) that selectively inhibits thimet oligopeptidase and neurolysin.
Pz peptidase A is an intracellular M3 metallopeptidase found in the thermophile Geobacillus collagenovorans MO-1 that recognizes collagen-specific tripeptide units (Gly-Pro-Xaa). Pz peptidase A shares common reactions with mammalian thimet oligopeptidase (TOP) and neurolysin, but has extremely low primary sequence identity to these enzymes. In this work, Pz peptidase A was cocrystallized with a phosphine peptide inhibitor (PPI) that selectively inhibits TOP and neurolysin. The crystals belong to space group P21, with unit-cell parameters a = 56.38, b = 194.15, c = 59.93 Å, β = 106.22°. This is the first crystallographic study of an M3 family peptidase–PPI complex.
doi:10.1107/S174430910700334X
PMCID: PMC2330125  PMID: 17277461
Pz peptidase A; M3 metallopeptidases; collagen degradation; Geobacillus collangenovorans MO-1
4.  Overexpression, purification, crystallization and preliminary X-ray cystallographic studies of a proline-specific aminopeptidase from Aneurinibacillus sp. strain AM-1 
Preliminary X-ray crystallographic study of a proline-specific aminopepitdase from Aneurinibacillus sp, strain AM-1 was carried out.
To elucidate the structure and molecular mechanism of a characteristic proline-specific aminopeptidase produced by the thermophile Aneurinibacillus sp. strain AM-1, its gene was cloned and the recombinant protein was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 1.8 Å resolution from the recombinant aminopeptidase crystal. The crystals belong to the orthorhombic space group P21212, with unit-cell parameters a = 93.62, b = 68.20, c = 76.84 Å. A complete data set was also obtained from crystals of SeMet-substituted aminopeptidase. Data in the resolution range 20–2.1 Å from the MAD data set from the SeMet-substituted crystal were used for phase determination.
doi:10.1107/S1744309106047543
PMCID: PMC2225360  PMID: 17142913
proline-specific aminopeptidase; Aneurinibacillus sp. strain AM-1; thermophiles
5.  A direct repeat of E-box-like elements is required for cell-autonomous circadian rhythm of clock genes 
Background
The circadian expression of the mammalian clock genes is based on transcriptional feedback loops. Two basic helix-loop-helix (bHLH) PAS (for Period-Arnt-Sim) domain-containing transcriptional activators, CLOCK and BMAL1, are known to regulate gene expression by interacting with a promoter element termed the E-box (CACGTG). The non-canonical E-boxes or E-box-like sequences have also been reported to be necessary for circadian oscillation.
Results
We report a new cis-element required for cell-autonomous circadian transcription of clock genes. This new element consists of a canonical E-box or a non-canonical E-box and an E-box-like sequence in tandem with the latter with a short interval, 6 base pairs, between them. We demonstrate that both E-box or E-box-like sequences are needed to generate cell-autonomous oscillation. We also verify that the spacing nucleotides with constant length between these 2 E-elements are crucial for robust oscillation. Furthermore, by in silico analysis we conclude that several clock and clock-controlled genes possess a direct repeat of the E-box-like elements in their promoter region.
Conclusion
We propose a novel possible mechanism regulated by double E-box-like elements, not to a single E-box, for circadian transcriptional oscillation. The direct repeat of the E-box-like elements identified in this study is the minimal required element for the generation of cell-autonomous transcriptional oscillation of clock and clock-controlled genes.
doi:10.1186/1471-2199-9-1
PMCID: PMC2254435  PMID: 18177499
6.  Crystallization and preliminary crystallographic analysis of bacterial fructosyl amino acid oxidase 
Crystallization of a fructosyl amino acid oxidase from Corynebacterium sp. 2-4-1 yields two forms: one monoclinic and one tetragonal.
Bacterial fructosyl amino acid oxidase [fructosyl α-l-amino acid:oxygen oxidoreductase (defructosylating); EC 1.5.3] has been crystallized by the hanging-drop vapour-diffusion technique using sodium citrate as the precipitant. Two types of crystals were grown: one type are rhombic prismatic yellow crystals that belong to space group C2 with unit-cell parameters a = 101.08, b = 63.36, c = 83.07 Å, β = 108.80° and diffract to at least 1.8 Å resolution, while the second type are rod-like crystals that belong to space group P4122 or P4322 with unit-cell parameters a = b = 119.09, c = 164.66 Å and diffract to 2.7 Å resolution.
doi:10.1107/S1744309104034372
PMCID: PMC1952257  PMID: 16510992
HbA1c; Amadori compound; fructosyl amino acid oxidase

Results 1-6 (6)