Crystal structure of Mycobacterium tuberculosis ketol-acid reductoisomerase at 1.0 Å resolution: A potential target for anti-tuberculosis drug discovery

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 angstrom resolution and the final model has an R-free of 0.163. The structure shows that the active site is solvent-accessible with the two metal ions separated by 4.7 angstrom. A comparison of this structure with that of Mg(-)(2+)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 angstrom are observed. This inherent flexibility therefore appears to be critical for initiation of the KARI catalyzed 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

Herbicides that target acetohydroxyacid synthase are potent inhibitors of the growth of drug resistant Candida auris

Acetohydroxyacid synthase (AHAS, EC 2.2.1.6) is the first enzyme in the branched chain amino acid biosynthesis pathway, is the target for more than 50 commercially available herbicides and is a promising target for antimicrobial drug discovery. Herein, we have expressed and purified AHAS from Candida auris, a newly identified human invasive fungal pathogen. Ten AHAS inhibiting herbicides have Ki values of 100 M and thus possesses a therapeutic index of >100. These data suggest that targeting AHAS is a viable strategy for treating C. auris infections

Engineering highly functional thermostable proteins using ancestral sequence reconstruction

Commercial biocatalysis requires robust enzymes that can withstand elevated temperatures and long incubations. Ancestral reconstruction has shown that pre-Cambrian enzymes were often much more thermostable than extant forms. Here, we resurrect ancestral enzymes that withstand similar to 30 degrees C higher temperatures and >= 100 times longer incubations than their extant forms. This is demonstrated on animal cytochromes P450 that stereo- and regioselectively functionalize unactivated C-H bonds for the synthesis of valuable chemicals, and bacterial ketol-acid reductoisomerases that are used to make butanol-based biofuels. The vertebrate CYP3 P450 ancestor showed a T-60(50) of 66 degrees C and enhanced solvent tolerance compared with the human drug-metabolizing CYP3A4, yet comparable activity towards a similarly broad range of substrates. The ancestral ketol-acid reductoisomerase showed an eight-fold higher specific activity than the cognate Escherichia coli form at 25 degrees C, which increased 3.5-fold at 50 degrees C. Thus, thermostable proteins can be devised using sequence data alone from even recent ancestors