来自普通脱硫弧菌宫崎F株的氢化酶单晶。

Single crystals of hydrogenase from Desulfovibrio vulgaris Miyazaki F.

作者信息

Higuchi Y, Yasuoka N, Kakudo M, Katsube Y, Yagi T, Inokuchi H

出版信息

J Biol Chem. 1987 Feb 25;262(6):2823-5.

Abstract

The hydrogenase solubilized from the particulate fraction from Desulfovibrio vulgaris Miyazaki F (IAM 12604) has been crystallized. Although the solubilized hydrogenase purified by the previous method (Yagi, T., Kimura, K., Daidoji, H., Sakai, F., Tamura, S., and Inokuchi, H. (1976) J. Biochem. (Tokyo) 79,661-671) revealed a single band upon disc electrophoresis, it could not be crystallized. The apparently homogeneous hydrogenase has been separated into three components of similar molecular weights by high performance liquid chromatography on DEAE-Toyopearl. Each hydrogenase component was successfully crystallized by means of the vapor diffusion method with polyethylene glycol or 2-methyl-2,4-pentanediol as a precipitating agent. Seeding procedure is necessary to grow an x-ray grade crystal. Preliminary x-ray experiments reveal that crystals grown from one component are in space group of P2(1)2(1)2(1) with a = 102.1(1), b = 126.8 (3), and c = 66.9(1) A. The unit cell volume of 8.66 X 10(5) A3 suggests that it contains one molecule/asymmetric unit (Vm = 2.43). The crystals grown from another component are in the same space group with a = 99.6(1), b = 126.8(3), c = 66.9(1) A, and the unit cell volume is 8.45 X 10(5) A3 (Vm = 2.37). The crystals diffract more than 2.5 A and are suitable for complete crystal analysis. Up to 4 A resolution native data have been collected on a diffractometer.

摘要

从普通脱硫弧菌宫崎F株（IAM 12604）的颗粒组分中溶解得到的氢化酶已被结晶。尽管通过先前方法（Yagi, T., Kimura, K., Daidoji, H., Sakai, F., Tamura, S., and Inokuchi, H. (1976) J. Biochem. (Tokyo) 79, 661 - 671）纯化得到的溶解氢化酶在圆盘电泳中显示出一条带，但它无法结晶。通过在DEAE - Toyopearl上进行高效液相色谱，已将明显均一的氢化酶分离为分子量相似的三个组分。每个氢化酶组分通过以聚乙二醇或2 - 甲基 - 2,4 - 戊二醇作为沉淀剂的气相扩散法成功结晶。生长X射线级晶体需要晶种程序。初步的X射线实验表明，从一个组分生长的晶体属于空间群P2(1)2(1)2(1)，a = 102.1(1)，b = 126.8 (3)，c = 66.9(1) Å。单位晶胞体积为8.66×10(5) Å3，表明其每个不对称单位包含一个分子（Vm = 2.43）。从另一个组分生长的晶体属于相同空间群，a = 99.6(1)，b = 126.8(3)，c = 66.9(1) Å，单位晶胞体积为8.45×10(5) Å3（Vm = 2.37）。这些晶体的衍射分辨率超过2.5 Å，适用于完整的晶体分析。已在衍射仪上收集了高达4 Å分辨率的天然数据。