title: Improved purification, crystallization and crystallographic study of Hyd-2-type [NiFe]-hydrogenase from Citrobacter sp. S-77.
abstract: The purification procedure of Hyd-2-type [NiFe]-hydrogenase from Citrobacter sp. S-77 was improved by applying treatment with trypsin before chromatography. Purified protein samples both with and without trypsin treatment were successfully crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol as a precipitant. Both crystals belonged to space group P21, with unit-cell parameters a = 63.90, b = 118.89, c = 96.70 Å, β = 100.61° for the protein subjected to trypsin treatment and a = 65.38, b = 121.45, c = 98.63 Å, β = 102.29° for the sample not treated with trypsin. The crystal obtained from the trypsin-treated protein diffracted to 1.60 Å resolution, which is considerably better than the 2.00 Å resolution obtained without trypsin treatment. The [NiFe]-hydrogenase from Citrobacter sp. S-77 retained catalytic activity with some amount of O2, indicating that it has clear O2 tolerance.
authors.affiliation: Department of Picobiology, University of Hyogo, 3-2-1 Koto, Kamigori-cho, Ako-gun, Hyogo 678-1297, Japan.
authors.forename: Noor Dina
authors.initials: ND
authors.lastname: Muhd Noor
authors.affiliation: Department of Picobiology, University of Hyogo, 3-2-1 Koto, Kamigori-cho, Ako-gun, Hyogo 678-1297, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Nishikawa
authors.affiliation: Department of Bioresource Science, Ibaraki University, 3-21-1 Chu-ou, Ami, Ibaraki 300-0393, Japan.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Nishihara
authors.affiliation: International Institute for Carbon-Neutral Energy Research (WPI-I2CNER), Kyushu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395, Japan.
authors.forename: Ki Seok
authors.initials: KS
authors.lastname: Yoon
authors.affiliation: Department of Chemistry and Biochemistry, Kyushu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395, Japan.
authors.forename: Seiji
authors.initials: S
authors.lastname: Ogo
authors.affiliation: Department of Picobiology, University of Hyogo, 3-2-1 Koto, Kamigori-cho, Ako-gun, Hyogo 678-1297, Japan.
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Higuchi
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D002954:Citrobacter / Q000201:enzymology*; D003460:Crystallization / Q000379:methods; D018360:Crystallography, X-Ray; D006859:Hydrogen / Q000737:chemistry; D006864:Hydrogenase / Q000737:chemistry / Q000302:isolation & purification*; D007700:Kinetics; D010100:Oxygen / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D006859:Hydrogen; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D010100:Oxygen
keywords: O2 tolerance; X-ray diffraction; [NiFe]-hydrogenase; crystallization