HYPB PROTEIN OF BRADYRHIZOBIUM-JAPONICUM IS A METAL-BINDING GTPASE CAPABLE OF BINDING 18 DIVALENT NICKEL IONS PER DIMER
HYPB PROTEIN OF BRADYRHIZOBIUM-JAPONICUM IS A METAL-BINDING GTPASE CAPABLE OF BINDING 18 DIVALENT NICKEL IONS PER DIMER
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DOI：
10.1073/pnas.92.6.2333
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发表时间：
1995-03-14
影响因子：
11.1
通讯作者：
MAIER, RJ
中科院分区：
文献类型：
--
作者：
FU, CL;OLSON, JW;MAIER, RJ
慢生型根瘤菌hiB编码一种含有极其丰富的组氨酸区域(在39个氨基酸范围内有24个组氨酸残基)和鸟嘌呤核苷酸结合区的蛋白质。该基因在大肠杆菌中高效表达，镍离子金属螯合亲和层析(MCAC)一步纯化。在SDS/PAGE中，HypB的迁移量为38 kDa，略大于计算的相对分子质量(32.8 kDa)。纯化的HypB具有GTPase活性，其K(CAT)为0.18min(-1)，GTP的K-m为7muM，也具有dGTPase活性。用Superose 12快速蛋白质液相色谱检测，HypB以分子量78 kDa的二聚体形式存在于天然溶液中。它结合了9.0+/-0.14个二价镍离子(每个二聚体18个Ni2+)，K-d为2.3u M；它还结合了锌、铜、钴、镉)和锰。HypB富含组氨酸区域的框内缺失(38个氨基酸缺失，包括23个组氨酸残基)导致截短的HypB不与MCAC柱结合，而HypB内14个氨基酸的缺失(包括8个组氨酸残基)导致截短的HypB仍然与该柱结合，结果表明HypB富含组氨酸区域内的组氨酸残基参与了金属结合。
Bradyrhizobium japonicum hypB encodes a protein containing an extremely histidine-rich region (24 histidine residues within a 39-amino-acid stretch) and guanine nucleotide-binding domains. The product of the hypB gene was overexpressed in Escherichia coli and purified by Ni2+-charged metal chelate affinity chromatography (MCAC) in a single step. In SDS/PAGE, HypB migrated at 38 kDa-slightly larger than the calculated molecular mass (32.8 kDa). Purified HypB has GTPase activity with a k(cat) of 0.18 min(-1) and a K-m for GTP of 7 mu M, and it has dGTPase activity as well. HypB exists as a dimer of molecular mass 78 kDa in native solution as determined by fast protein liquid chromatography on Superose 12. It binds 9.0 +/- 0.14 divalent nickel ions per monomer (18 Ni2+ per dimer) with a K-d of 2.3 mu M; it also binds Zn2+, CU2+, Co2+, Cd2+) and Mn2+. In-frame deletion of the histidine rich region (deletion of 38 amino acids including 23 histidine residues) resulted in a truncated HypB that did not bind to the MCAC column, whereas in-frame deletion of 14 amino acids including 8 histidine residues within HypB resulted in a truncated HypB that still bound to the column, The results indicate that the histidine residues within the histidine-rich region of HypB are involved in metal binding.