Essential roles of nucleotide-switch and metal-coordinating residues for chaperone function of diol dehydratase-reactivase

Koichi Mori, Koji Obayashi, Yasuhiro Hosokawa, Akina Yamamoto, Mayumi Yano, Toshiyuki Yoshinaga, Tetsuo Toraya

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

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 Mg 2+-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 Mg 2+ is essential for subunit swapping and therefore for (re)activation.

Original languageEnglish
Pages (from-to)8677-8686
Number of pages10
JournalBiochemistry
Volume52
Issue number48
DOIs
Publication statusPublished - Dec 3 2013

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Propanediol Dehydratase
Hydro-Lyases
Nucleotides
Metals
Switches
Adenosine Triphosphate
Vitamin B 12
Adenosine Diphosphate
Adenosine Triphosphatases
Binding Sites
Molecular Chaperones

ASJC Scopus subject areas

  • Biochemistry

Cite this

Essential roles of nucleotide-switch and metal-coordinating residues for chaperone function of diol dehydratase-reactivase. / Mori, Koichi; Obayashi, Koji; Hosokawa, Yasuhiro; Yamamoto, Akina; Yano, Mayumi; Yoshinaga, Toshiyuki; Toraya, Tetsuo.

In: Biochemistry, Vol. 52, No. 48, 03.12.2013, p. 8677-8686.

Research output: Contribution to journalArticle

Mori, K, Obayashi, K, Hosokawa, Y, Yamamoto, A, Yano, M, Yoshinaga, T & Toraya, T 2013, 'Essential roles of nucleotide-switch and metal-coordinating residues for chaperone function of diol dehydratase-reactivase', Biochemistry, vol. 52, no. 48, pp. 8677-8686. https://doi.org/10.1021/bi401290j
Mori, Koichi ; Obayashi, Koji ; Hosokawa, Yasuhiro ; Yamamoto, Akina ; Yano, Mayumi ; Yoshinaga, Toshiyuki ; Toraya, Tetsuo. / Essential roles of nucleotide-switch and metal-coordinating residues for chaperone function of diol dehydratase-reactivase. In: Biochemistry. 2013 ; Vol. 52, No. 48. pp. 8677-8686.
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abstract = "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 Mg 2+-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 Mg 2+ is essential for subunit swapping and therefore for (re)activation.",
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AU - Mori, Koichi

AU - Obayashi, Koji

AU - Hosokawa, Yasuhiro

AU - Yamamoto, Akina

AU - Yano, Mayumi

AU - Yoshinaga, Toshiyuki

AU - Toraya, Tetsuo

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N2 - 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 Mg 2+-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 Mg 2+ is essential for subunit swapping and therefore for (re)activation.

AB - 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 Mg 2+-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 Mg 2+ is essential for subunit swapping and therefore for (re)activation.

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