Use of Cryo-EM To Uncover Structural Bases of pH Effect and Cofactor Bispecificity of Ketol-Acid Reductoisomerase.
J Am Chem Soc
; 141(15): 6136-6140, 2019 04 17.
Article
em En
| MEDLINE
| ID: mdl-30921515
ABSTRACT
While cryo-EM is revolutionizing structural biology, its impact on enzymology is yet to be fully demonstrated. The ketol-acid reductoisomerase (KARI) catalyzes conversion of (2 S)-acetolactate or (2 S)-aceto-2-hydroxybutyrate to 2,3-dihydroxy-3-alkylbutyrate. We found that KARI from archaea Sulfolobus solfataricus (Sso-KARI) is unusual in being a dodecamer, bispecific to NADH and NADPH, and losing activity above pH 7.8. While crystals were obtainable only at pH 8.5, cryo-EM structures were solved at pH 7.5 and 8.5 for Sso-KARI2Mg2+. The results showed that the distances of the two catalytic Mg2+ ions are lengthened in both structures at pH 8.5. We next solved cryo-EM structures of two Sso-KARI complexes, with NADH+inhibitor and NADPH+inhibitor at pH 7.5, which indicate that the bispecificity can be attributed to a unique asparagine at the cofactor binding loop. Unexpectedly, Sso-KARI also differs from other KARI enzymes in lacking "induced-fit", reflecting structural rigidity. Thus, cryo-EM is powerful for structural and mechanistic enzymology.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Archaea
/
Álcoois
/
Cetol-Ácido Redutoisomerase
/
Cetonas
Idioma:
En
Revista:
J Am Chem Soc
Ano de publicação:
2019
Tipo de documento:
Article
País de afiliação:
Taiwan