EMD-9801
Cryo-EM Structure of Sulfolobus solfataricus ketol-acid reductoisomerase (Sso-KARI) in complex with Mg2+, NADH, and CPD at pH7.5
EMD-9801
Single-particle3.38 Å
Deposition: 30/01/2019Map released: 17/04/2019
Last modified: 27/03/2024
Sample Organism:
Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2)
Sample: Cryo-EM Structure of Sulfolobus solfataricus ketol-acid reductoisomerase (Sso-KARI) dodecamer in complex with Mg2+, NADH, and CPD at pH7.5
Fitted models: 6jd1 (Avg. Q-score: 0.351)
Deposition Authors: Chen CY, Chang YC
,
Lin KF ,
Huang CH,
Lin BL
Sample: Cryo-EM Structure of Sulfolobus solfataricus ketol-acid reductoisomerase (Sso-KARI) dodecamer in complex with Mg2+, NADH, and CPD at pH7.5
Fitted models: 6jd1 (Avg. Q-score: 0.351)
Deposition Authors: Chen CY, Chang YC
,
Lin KF ,
Huang CH,
Lin BL
Use of Cryo-EM To Uncover Structural Bases of pH Effect and Cofactor Bispecificity of Ketol-Acid Reductoisomerase.
Chen CY,
Chang YC ,
Lin BL,
Lin KF ,
Huang CH,
Hsieh DL,
Ko TP ,
Tsai MD
(2019) J Am Chem Soc , 141 , 6136 - 6140
,
Lin BL,
Lin KF ,
Huang CH,
Hsieh DL,
Ko TP ,
Tsai MD
(2019) J Am Chem Soc , 141 , 6136 - 6140
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-KARI:2Mg2+. 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.
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-KARI:2Mg2+. 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.