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The branched-chain amino acids (BCAAs) leucine, isoleucine and valine are synthesized via a common biosynthetic pathway. Ketol-acid reductoisomerase (KARI) is the second enzyme in this pathway. In addition to its role in BCAA biosynthesis, KARI catalyzes two rate-limiting steps that are key components of a cell-free biofuel biosynthesis route. For industrial applications, reaction temperature and enzyme stability are key factors that affect process robustness and product yield. Here, we have solved the cryo-EM structure (2.94 Å resolution) of a homododecameric Class I KARI (from Campylobacter jejuni) and demonstrated how a triad of amino acid side chains plays a crucial role in promoting the oligomerization of this enzyme. Importantly, both its thermal and solvent stability are greatly enhanced in the dodecameric state when compared to its dimeric counterpart (apparent melting temperatures (Tm) of 83.1 °C and 51.5 °C, respectively). We also employed protein design (PROSS) for a tetrameric Class II KARI (from Escherichia coli) to generate a variant with improved thermal and solvent stabilities. In total, 34 mutations were introduced, which did not affect the oligomeric state of this enzyme but resulted in a fully functional catalyst with a significantly elevated Tm (58.5 °C vs. 47.9 °C for the native version). [ABSTRACT FROM AUTHOR]

The biosynthetic pathway for the branched-chain amino acids is present in plants, fungi and bacteria, but not in animals, making it an attractive target for herbicidal and antimicrobial drug discovery. Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) is the second enzyme in this pathway, converting in a Mg2+- and NADPH-dependent reaction either 2-acetolactate or 2-aceto-2-hydroxybutyrate to their corresponding 2,3-dihydroxy-3- alkylbutyrate products. Here, we have determined the crystal structure of Mycobacterium tuberculosis (Mt) KARI, a class I KARI, with two magnesium ions bound in the active site. X-ray data were obtained to 1.0 Å resolution and the final model has an Rfree of 0.163. The structure shows that the active site is solvent-accessible with the two metal ions separated by 4.7 Å. A comparison of this structure with that of Mg2+-free Pseudomonas aeruginosa KARI suggests that upon magnesium binding no movement of the N domain relative to the C domain occurs. However, upon formation of the Michaelis complex, as illustrated in the structure of Slackia exigua KARI in complex with NADH.Mg2+. N-hydroxy-N-isopropyloxamate (IpOHA, a transition state analog), domain movements and reduction of the metal-metal distance to 3.5 Å are observed. This inherent flexibility therefore appears to be critical for initiation of the KARIcatalyzed reaction. This study provides new insights into the complex structural rearrangements required for activity of KARIs, particularly those belonging to class I, and provides the framework for the rational design of Mt KARI inhibitors that can be tested as novel antituberculosis agents. [ABSTRACT FROM AUTHOR]