Essential roles of nucleotide-switch and metal-coordinating residues for chaperone function of diol dehydratase-reactivase.
Essential roles of nucleotide-switch and metal-coordinating residues for chaperone function of diol dehydratase-reactivase.
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核苷酸开关和金属配位残基对于二醇脱水酶重激活酶的伴侣功能的重要作用。
DOI：
10.1021/bi401290j
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发表时间：
2013
期刊：
影响因子：
2.9
通讯作者：
and Tetsuo Toraya
中科院分区：
文献类型：
--
作者：
Koichi Mori;Koji Obayashi;Yasuhiro Hosokawa;Akina Yamamoto;Mayumi Yano;Toshiyuki Yoshinaga;and Tetsuo Toraya
二醇脱氢酶-再活化酶（DD-R）是一种分子伴侣，通过辅因子交换使失活的全二醇脱氢酶（DD）再活化。其ADP结合形式和ATP结合形式分别是DD的高亲和力和低亲和力形式。在核苷酸结合位点突变的DD-R中，Dα 8 N和Dα 413 N突变体都不能作为有效的再活化酶。虽然Dα 413 N具有ATP酶活性，但在ATP或ADP存在下，Dα 413 N不介导氰钴胺（CN Cbl）从DD·CN Cbl复合物中释放，即使在ATP存在下，D α 413 N也与apoDD形成紧密复合物，提示Aspα413参与了核苷酸开关。与此相反，Dα 8 N显示出非常低的ATP酶活性，并且在ATP存在下不介导CN-Cbl从复合物中释放，但在ADP存在下它确实引起约50%的释放。该突变体与DD形成的复合物被ATP部分逆转，表明Aspα8参与ATP酶活性，但仅部分参与核苷酸开关。在Mg 2+结合位点突变的DD-R中，只有Eβ 31 Q的活性约为野生型DD-R的30%，并与apoDD形成紧密的复合物，表明DD-R β亚基不是重新激活所必需的。如果DD β和DD R β之间发生亚基交换，则DD的Gluβ97将与Mg 2+配位。Eβ 97 Q DD与CN-Cbl的复合物不被野生型DD-R激活。该突变体和野生型DD-R之间没有形成复合物，表明Gluβ97与Mg 2+的配位对于亚基交换以及因此对于（再）激活是必需的。
Diol dehydratase-reactivase (DD-R) is a molecular chaperone that reactivates inactivated holodiol dehydratase (DD) by cofactor exchange. Its ADP-bound and ATP-bound forms are high-affinity and low-affinity forms for DD, respectively. Among DD-Rs mutated at the nucleotide-binding site, neither the Dα8N nor Dα413N mutant was effective as a reactivase. Although Dα413N showed ATPase activity, it did not mediate cyanocobalamin (CN-Cbl) release from the DD·CN-Cbl complex in the presence of ATP or ADP and formed a tight complex with apoDD even in the presence of ATP, suggesting the involvement of Aspα413 in the nucleotide switch. In contrast, Dα8N showed very low ATPase activity and did not mediate CN-Cbl release from the complex in the presence of ATP, but it did cause about 50% release in the presence of ADP. The complex formation of this mutant with DD was partially reversed by ATP, suggesting that Aspα8 is involved in the ATPase activity but only partially in the nucleotide switch. Among DD-Rs mutated at the Mg2+-binding site, only Eβ31Q was about 30% as active as wild-type DD-R and formed a tight complex with apoDD, indicating that the DD-R β subunit is not absolutely required for reactivation. If subunit swapping occurs between the DD-R β and DD β subunits, Gluβ97 of DD would coordinate to Mg2+. The complex of Eβ97Q DD with CN-Cbl was not activated by wild-type DD-R. No complex was formed between this mutant and wild-type DD-R, indicating that the coordination of Gluβ97 to Mg2+is essential for subunit swapping and therefore for (re)activation.