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 language | English |
|---|---|
| Pages (from-to) | 8677-8686 |
| Number of pages | 10 |
| Journal | Biochemistry |
| Volume | 52 |
| Issue number | 48 |
| DOIs | |
| Publication status | Published - Dec 3 2013 |
Fingerprint
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 journal › Article
}
TY - JOUR
T1 - Essential roles of nucleotide-switch and metal-coordinating residues for chaperone function of diol dehydratase-reactivase
AU - Mori, Koichi
AU - Obayashi, Koji
AU - Hosokawa, Yasuhiro
AU - Yamamoto, Akina
AU - Yano, Mayumi
AU - Yoshinaga, Toshiyuki
AU - Toraya, Tetsuo
PY - 2013/12/3
Y1 - 2013/12/3
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.
UR - http://www.scopus.com/inward/record.url?scp=84889255792&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84889255792&partnerID=8YFLogxK
U2 - 10.1021/bi401290j
DO - 10.1021/bi401290j
M3 - Article
AN - SCOPUS:84889255792
VL - 52
SP - 8677
EP - 8686
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 48
ER -