Use of Cryo-EM To Uncover Structural Bases of pH Effect and Cofactor Bispecificity of Ketol-Acid Reductoisomerase

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 (2S)-acetolactate or (2S)-aceto-2hydroxybutyrate 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:2Mg 2+ . The results showed that the distances of the two catalytic Mg 2+ 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.

2.7 Å cryo-EM structure of vitrified M. musculus H-chain apoferritin from 200 keV “screening microscope”

“…Such sophisticated equipment is however expensive, requiring high-level strategies to maintain microscope stability and performance, so that the method would appear to be accessible in a daily manner to only few laboratories. Recent publications have explored the potential of 200 kV microscope to obtain reconstructions higher that 3 Å [13], [14] [15], [16] and [17], see Table S2. The dedicated instrument used to resolve those molecular structures is, again, highly sophisticated, and is in addition, costly.…”

Structural Rearrangements of a Dodecameric Ketol-Acid Reductoisomerase Isolated from a Marine Thermophilic Methanogen

“…Numerous structures of class I KARIs were obtained with Mg 2+ ions, NAD(P)H, substrate analogues and/or inhibitors [6][7][8][10][11][12][13]16,17,20]. This deep and collective work highlighted some structural rearrangements triggered by binding of Mg 2+ and NAD(P)H [12,13,17,20].…”

“…KARIs are structurally composed of an N-terminal Rossmann domain and a Cterminal "knot" domain [1,7]. They can be distinguished in two classes: the short class I gathering dimeric and dodecameric enzymes, and the monomeric or tetrameric longer class II KARIs [1,[6][7][8][9][10][11][12][13][14]. Prokaryotes can harbour class I or II enzymes, while eukaryotes only contain class II.…”

“…Most KARIs are dependent on NADPH, which was proposed to be the ancestral trait [1,6,15,16]. However, NADH-dependent or bispecific enzymes were also discovered [8,13,[15][16][17]. The use of NADH-dependent enzymes is preferred for biotechnological processes since the cellular content of NADH is higher than NADPH, a ratio that would stimulate the amino acid production rate.…”

2.7 Å cryo-EM structure of vitrified M. musculus H-chain apoferritin from a compact 200 keV cryo-microscope

“…Such sophisticated equipment is however expensive, requiring high-level strategies to maintain microscope stability and performance, so that the method would appear to be inaccessible on a daily basis to many laboratories. Recent publications have explored the potential of 200 kV microscopes to obtain reconstructions higher than 3 Å [15][16][17][18][19], see S2 Table. With the exception of one group that uses a JEOL cryo-ARM 1 microscope with an in-column energy filter and a sophisticated projection system, data for each of these have been collected using instruments of the Arctica 1 microscope series, which includes a two-lens condenser system. Such dedicated instruments are costly and have a rather large footprint.…”