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.
Alcohols/metabolism , Archaea/enzymology , Ketol-Acid Reductoisomerase/chemistry , Ketones/metabolism , Alcohols/chemistry , Crystallography, X-Ray , Hydrogen-Ion Concentration , Ketol-Acid Reductoisomerase/metabolism , Ketones/chemistry , Models, Molecular , Molecular Conformation
Programmed cell death 5 (PDCD5) is a vital signaling protein in the apoptosis pathway in eukaryotes. It is known that there are two dissociated N-terminal regions and a triple-helix core in eukaryotic PDCD5. Structural and functional studies of PDCD5 from hyperthermophilic archaea have been limited to date. Here, the PDCD5 homolog Sso0352 (SsoPDCD5) was identified in Sulfolobus solfataricus, the SsoPDCD5 protein was expressed and crystallized, and the phase was identified by single-wavelength anomalous diffraction. The native SsoPDCD5 crystal belonged to space group C2 and diffracted to 1.49â Å resolution. This is the first crystal structure of a PDCD5 homolog to be solved. SsoPDCD5 shares a similar triple-helix bundle with eukaryotic PDCD5 but has a long α-helix in the N-terminus. A structural search and biochemical data suggest that SsoPDCD5 may function as a DNA-binding protein.
Apoptosis Regulatory Proteins/chemistry , Neoplasm Proteins/chemistry , Sulfolobus solfataricus/enzymology , Amino Acid Sequence , Apoptosis Regulatory Proteins/isolation & purification , Apoptosis Regulatory Proteins/metabolism , Crystallization , Crystallography, X-Ray , Humans , Models, Molecular , Neoplasm Proteins/isolation & purification , Neoplasm Proteins/metabolism , Protein Binding , Protein Conformation , Sequence Homology
