Komeda H, Kobayashi M, Shimizu S
Department of Agricultural Chemistry, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Japan.
Proc Natl Acad Sci U S A. 1997 Jan 7;94(1):36-41. doi: 10.1073/pnas.94.1.36.
Cobalt is an essential component of a low molecular-mass nitrile hydratase (L-NHase) from Rhodococcus rhodochrous J1. We have found a new gene, nhlF, in the DNA region sandwiched between nhlBA encoding L-NHase and amdA encoding amidase, which are involved in the degradation of nitriles. The product of nhlF, NhlF, shows a significant sequence similarity with those of hoxN from Alcaligenes eutrophus, hupN from Bradyrhizobium japonicum, nixA from Helicobacter pylori, and ureH from Bacillus sp., which are considered to be involved in nickel uptake into these cells. Sequence and hydropathy plot analyses have shown that NhlF encodes a 352-amino acid (aa) protein with eight hydrophobic putative membrane-spanning domains. nhlF expression in R. rhodochrous ATCC 12674 and Escherichia coli JM109 confers uptake of 57Co in their cells, but not of 63Ni. The expression of both nhlF and nhlBA in R. rhodochrous ATCC 12674 exhibited higher NHase activity than nhlBA expression. These findings together with the inhibitory effect by uncouplers (CCCP and SF6847) for the cobalt uptake suggest that NhlF mediates the cobalt transport into the cell energy-dependently finally to provide L-NHase.
钴是来自红平红球菌J1的低分子量腈水合酶（L-NHase）的必需成分。我们在编码L-NHase的nhlBA和编码腈水解酶的amdA之间的DNA区域发现了一个新基因nhlF，它们都参与腈的降解。nhlF的产物NhlF与嗜碱产碱杆菌的hoxN、日本慢生根瘤菌的hupN、幽门螺杆菌的nixA以及芽孢杆菌属的ureH的产物具有显著的序列相似性，这些蛋白被认为参与这些细胞对镍的摄取。序列分析和亲水性图谱分析表明，NhlF编码一个含有8个假定跨膜疏水结构域的352个氨基酸（aa）的蛋白质。nhlF在红平红球菌ATCC 12674和大肠杆菌JM109中的表达使它们的细胞能够摄取57Co，但不能摄取63Ni。nhlF和nhlBA在红平红球菌ATCC 12674中的共同表达比nhlBA单独表达表现出更高的NHase活性。这些发现以及解偶联剂（CCCP和SF6847）对钴摄取的抑制作用表明，NhlF以能量依赖的方式介导钴进入细胞，最终为L-NHase提供钴。
