Substrate-Specific Differences in Catalytic Strategy and Activity across Ketol-Acid Reductoisomerase Variants

The specificity and catalytic efficiency of enzymes make them attractive for applications ranging from therapeutics to chemical manufacturing. However, it remains challenging to identify specific structural and dynamic mechanisms by which enzymes achieve their catalytic rate enhancements as well as to re-engineer enzymes to improve their catalytic properties. In earlier work we reported the design and selection of KARI mutants with calculated increases in specific activity (i.e., kcat) relative to wild type (WT) for the isomerization step of one of its native substrates: 2-acetolactate (ACL), which leads to the synthesis of the amino acids valine and leucine. Eight mutants were identified with computed improvements in kcat of up to 4 orders of magnitude. In the current study, we investigate the effects of these same mutations on the isomerization of the other native substrate, 2-aceto-2-hydroxybutyrate (AHB, leading to the synthesis of isoleucine). Paralleling our previous work, we use the computational statistical mechanical method transition interface sampling (TIS) to simulate reaction kinetics and compute reaction rate constants. We find that the mutants selected for increased efficiency on ACL had varied levels of activity on AHB-some enhancing reactivity and others diminishing it-with the range in computed AHB rate constants spanning more than 7 orders of magnitude. Analysis of the simulations for WT-AHB revealed that only some of the structural mechanisms associated with mutants' improved ACL catalysis were expected to transfer to, and thereby improve, AHB catalysis. For two mutants with significantly lower catalytic efficiency on AHB than WT, further analysis identified unique conformational changes that may explain their low activity on AHB.