嗜热栖热氢单胞菌TH-1的NAD⁺还原型[NiFe]氢化酶的结晶及初步X射线分析
Crystallization and preliminary X-ray analysis of the NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus TH-1.
作者信息
Taketa Midori, Nakagawa Hanae, Habukawa Mao, Osuka Hisao, Kihira Kiyohito, Komori Hirofumi, Shibata Naoki, Ishii Masaharu, Igarashi Yasuo, Nishihara Hirofumi, Yoon Ki-Seok, Ogo Seiji, Shomura Yasuhito, Higuchi Yoshiki
机构信息
Department of Picobiology, University of Hyogo, 3-2-1 Koto, Kamigori-cho, Ako-gun, Hyogo 678-1297, Japan.
Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
出版信息
Acta Crystallogr F Struct Biol Commun. 2015 Jan 1;71(Pt 1):96-9. doi: 10.1107/S2053230X14026521.
NAD+-reducing [NiFe] hydrogenases catalyze the oxidoreduction of dihydrogen concomitant with the interconversion of NAD+ and NADH. Here, the isolation, purification and crystallization of the NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus TH-1 are reported. Crystals of the NAD+-reducing [NiFe] hydrogenase were obtained within one week from a solution containing polyethylene glycol using the sitting-drop vapour-diffusion method and micro-seeding. The crystal diffracted to 2.58 Å resolution and belonged to space group C2, with unit-cell parameters a=131.43, b=189.71, c=124.59 Å, β=109.42°. Assuming the presence of two NAD+-reducing [NiFe] hydrogenase molecules in the asymmetric unit, VM was calculated to be 2.2 Å3 Da(-1), which corresponds to a solvent content of 43%. Initial phases were determined by the single-wavelength anomalous dispersion method using the anomalous signal from the Fe atoms.
NAD⁺还原型[NiFe]氢化酶催化二氢的氧化还原反应，同时伴随着NAD⁺和NADH的相互转化。本文报道了嗜热栖热氢菌TH-1中NAD⁺还原型[NiFe]氢化酶的分离、纯化及结晶过程。采用坐滴气相扩散法并结合微量接种，在一周内从含有聚乙二醇的溶液中获得了NAD⁺还原型[NiFe]氢化酶的晶体。该晶体的衍射分辨率为2.58 Å，属于空间群C2，晶胞参数a = 131.43、b = 189.71、c = 124.59 Å，β = 109.42°。假设不对称单元中存在两个NAD⁺还原型[NiFe]氢化酶分子，计算出的VM为2.2 ų Da⁻¹，对应的溶剂含量为43%。利用Fe原子的异常信号，通过单波长异常色散法确定了初始相位。
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