Karst Johanna C, Foucher Anne-Emmanuelle, Campbell Tracey L, Di Guilmi Anne-Marie, Stroebel David, Mangat Chand S, Brown Eric D, Jault Jean-Michel
Institut de Biologie Structurale, UMR 5075 Université Joseph Fourier/CEA/CNRS, 41 rue Jules Horowitz, 38027 Grenoble cedex 1, France.
Antimicrobial Research Centre, Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main Street West, Hamilton, ON L8N 3Z5, Canada.
Microbiology (Reading). 2009 Mar;155(Pt 3):944-956. doi: 10.1099/mic.0.021543-0.
Characterization of 'unknown' proteins is one of the challenges of the post-genomic era. Here, we report a study of Bacillus subtilis YdiB, which belongs to an uncharted class of bacterial P-loop ATPases. Precise deletion of the ydiB gene yielded a mutant with much reduced growth rate compared to the wild-type strain. In vitro, purified YdiB was in equilibrium among different forms, monomers, dimers and oligomers, and this equilibrium was strongly affected by salts; high concentrations of NaCl favoured the monomeric over the oligomeric form of the enzyme. Interestingly, the ATPase activity of the monomer was about three times higher than that of the oligomer, and the monomer showed a K(m) of about 60 microM for ATP and a V(max) of about 10 nmol min(-1) (mg protein)(-1) (k(cat) approximately 10 h(-1)). This low ATPase activity was shown to be specific to YdiB because mutation of an invariant lysine residue in the P-loop motif (K41A) strongly attenuated this rate. This mutant was unable to restore a normal growth phenotype when introduced into a conditional knockout strain for ydiB, showing that the ATPase activity of YdiB is required for the in vivo function of the protein. Oligomerization was also observed with the purified YjeE from Escherichia coli, a YdiB orthologue, suggesting that this property is shared by all members of this family of ATPases. Importantly, dimers of YdiB were also observed in a B. subtilis extract, or when stabilized by formaldehyde cross-linking for YjeE from E. coli, suggesting that oligomerization might regulate the function of this new class of proteins in vivo.
“未知”蛋白质的特性鉴定是后基因组时代的挑战之一。在此，我们报道了一项关于枯草芽孢杆菌YdiB的研究，它属于一类尚未明确的细菌P环ATP酶。与野生型菌株相比，ydiB基因的精确缺失产生了一个生长速率大幅降低的突变体。在体外，纯化的YdiB在单体、二聚体和寡聚体等不同形式之间处于平衡状态，且这种平衡受到盐的强烈影响；高浓度的NaCl有利于酶的单体形式而非寡聚体形式。有趣的是，单体的ATP酶活性约为寡聚体的三倍，单体对ATP的K(m)约为60微摩尔，V(max)约为10纳摩尔每分钟（毫克蛋白）（-1）（k(cat)约为10小时（-1））。这种低ATP酶活性被证明是YdiB所特有的，因为P环基序中一个不变的赖氨酸残基（K41A）发生突变会强烈降低该速率。当将此突变体导入ydiB的条件性敲除菌株时，无法恢复正常的生长表型，这表明YdiB的ATP酶活性是该蛋白体内功能所必需的。从大肠杆菌中纯化的YdiB直系同源物YjeE也观察到了寡聚化现象，这表明该ATP酶家族的所有成员都具有这一特性。重要的是，在枯草芽孢杆菌提取物中也观察到了YdiB的二聚体，或者当通过甲醛交联使大肠杆菌的YjeE稳定时也观察到了，这表明寡聚化可能在体内调节这类新蛋白质的功能。
