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  • [Crystallization] Preliminary diffraction analysis of Salmonella Typhi YfdX crystallized in the dimeric state under high pH conditions

    YfdX is a bacterial protein that has been identified in a number of gram-negative species, including Salmonella enterica serovar Typhi, which is the causative agent of typhoid fever. Previously, we reported the pH-dependent change of the oligomeric state of S. Typhi YfdX between tetrameric (at pH 5.5) and dimeric (at pH 8.0) forms, which was demonstrated by size exclusion chromatography-multiangle light scattering and small angle X-ray scattering. However, only the threedimensional structure of tetrameric YfdX determined using crystals obtained under low pH conditions was presented in our previous study. In this study, in an effort to obtain structural information for YfdX in the dimeric state at high pH, wildtype and Sel-Met-substituted recombinant S. Typhi YfdX proteins were produced in an Escherichia coli expression system, purified using Ni-NTA affinity chromatography and size exclusion chromatography, and crystallized. Crystals obtained at high pH were selected for further optimization to determine the crystal structure of dimeric YfdX. X-ray diffraction data were collected at resolutions of 1.70 Å and 1.90 Å for native and Sel-Met-substituted crystals, respectively. A preliminary diffraction analysis was conducted, which indicated that our crystals commonly belong to the P2 space group, with six YfdX molecules present in the single asymmetric unit.

  • [Crystallization] Cytosolic 5′-Methylthioadenosine/S-Adenosylhomocysteine Nucleosidase MtaN from Mycobacterium. smegmatis: purification, crystallization and X-ray crystallographic analysis

    Mycobacterium tuberculosis is a dangerous pathogen, and it can cause the most deadly disease tuberculosis (TB). Nonpathogenic Mycobacterium smegmatis is an important model for studying the M. tuberculosis. M. smegmatis 5’-Methylthioadenosine/S-adenosyl-L-homocysteine nucleosidases (MtaNs) catalyze the hydrolysis of adenine from 5’-methylthioadenosine (MTA), MtaNs cleave the glycosidic bond of MTA or S-adenosylhomocysteine (SAH) irreversibly. In this study, MtaN from M. smegmatis (MsMtaN) was successfully expressed and purified using Ni-NTA affinity, Q anionexchange and gel-filtration chromatography. The protein crystal was obtained and diffracted to a resolution of 2.0 Å. The crystal belonged to the orthorhombic space group P1211, with unit-cell parameters of a =57.6, b = 172.6, and c = 183.3 Å. The Matthews coefficient and solvent content were estimated to be 2.32 Å3 Da-1 and 47%, respectively, assuming that the asymmetric unit contained only one recombinant protein molecule. Size-exclusion chromatography suggested that MsMtaN prefer to exist as tetramer in solution.

  • [Crystallization] Purification, Crystallization and X-ray crystallographic analysis of Homoserine O-succinyltransferase from Escherichia coli

    Homoserine O-succinyltransferase from Escherichia coli (EcHST) is the first enzyme in methionine biosynthesis and it catalyzed the transfer of succinyl-group from succinyl-CoA to L-homoserine. EcHST has reported to be regulated by feedback inhibition and it controls the bacterial growth due to its instability. To improve the EcHST for industrial application, the protein was overexpressed and purified to homogeneity by Ni-NTA affinity and size-exclusion chromatography. The EcHST protein was crystallized using hanging-drop vapor-diffusion method in the presence of 25% polyethylene glycol 3350, 0.1 M HEPES, pH 7.5, and 0.1 M magnesium chloride at 295 K. X-ray diffraction data were collected to a maximum resolution of 1.7 Å. The EcHST crystals belonged to the space group P212121 with unit cell parameters a = 76.35 Å, b = 81.03 Å, c = 98.09, α = β = γ = 90.0°. With two molecules of EcHST per asymmetric unit, the crystal volume per unit of protein mass was 2.12 Å3 Da-1, which correspond to a solvent content was approximately 42.15%.