Integrated Center for Sciences (INCS), Ehime University, Matsuyama, Japan.
J Bacteriol. 2012 Sep;194(17):4546-9. doi: 10.1128/JB.00714-12. Epub 2012 Jun 22.
The crystal structures of the Na(+)- and Li(+)-bound NtpK rings of Enterococcus hirae V-ATPase have been obtained. The coupling ion (Na(+) or Li(+)) was surrounded by five oxygen atoms contributed by residues T64, Q65, Q110, E139, and L61, and the hydrogen bonds of the side chains of Q110, Y68, and T64 stabilized the position of the E139 γ carboxylate essential for ion occlusion (PDB accession numbers 2BL2 and 2CYD). We previously indicated that an NtpK mutant strain (E139D) lost tolerance to sodium but not to lithium at alkaline pHs and suggested that the E139 residue is indispensable for the enzymatic activity of E. hirae V-ATPase linked with the sodium tolerance of this bacterium. In this study, we examined the activities of V-ATPase in which these four residues, except for E139, were substituted. The V-ATPase activities of the Q65A and Y68A mutants were slightly retained, but those of the T64A and Q110A mutants were negligible. Among the residues, T64 and Q110 are indispensable for the ion coupling of E. hirae V-ATPase, in addition to the essential residue E139.
已获得来自屎肠球菌 V-ATP 酶的 Na(+)-和 Li(+)-结合 NtpK 环的晶体结构。偶联离子（Na(+) 或 Li(+)）被由残基 T64、Q65、Q110、E139 和 L61 贡献的五个氧原子包围，侧链 Q110、Y68 和 T64 的氢键稳定了 E139 γ 羧酸盐的位置，这对于离子封闭至关重要（PDB 访问号 2BL2 和 2CYD）。我们之前指出，NtpK 突变株（E139D）在碱性 pH 值下失去了对钠离子的耐受性，但对锂离子没有耐受性，并表明 E139 残基对于与该细菌的钠离子耐受性相关的屎肠球菌 V-ATP 酶的酶活性是不可或缺的。在这项研究中，我们检查了除 E139 以外的这四个残基被取代的 V-ATP 酶的活性。Q65A 和 Y68A 突变体的 V-ATP 酶活性略有保留，但 T64A 和 Q110A 突变体的 V-ATP 酶活性几乎可以忽略不计。除了必需残基 E139 之外，T64 和 Q110 对于屎肠球菌 V-ATP 酶的离子偶联也是不可或缺的。
