Essential roles of nucleotide-switch and metal-coordinating residues for chaperone function of diol dehydratase-reactivase.
Biochemistry
; 52(48): 8677-86, 2013 Dec 03.
Article
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| MEDLINE
| ID: mdl-24229359
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 Eß31Q was about 30% as active as wild-type DD-R and formed a tight complex with apoDD, indicating that the DD-R ß subunit is not absolutely required for reactivation. If subunit swapping occurs between the DD-R ß and DD ß subunits, Gluß97 of DD would coordinate to Mg(2+). The complex of Eß97Q 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 Gluß97 to Mg(2+) is essential for subunit swapping and therefore for (re)activation.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Propanodiol Deshidratasa
/
Chaperonas Moleculares
/
Nucleótidos
Límite:
Humans
Idioma:
En
Revista:
Biochemistry
Año:
2013
Tipo del documento:
Article
País de afiliación:
Japón
Pais de publicación:
Estados Unidos