Mapping of the Reaction Trajectory Catalyzed by Class I Ketol-Acid Reductoisomerase

异构化 催化作用 活动站点 催化循环 立体化学 化学 酶催化 反应速率 QM/毫米 生物化学
作者
Xin Lin,Thierry Lonhienne,You Lv,Julia L. Kurz,Ross P. McGeary,Gerhard Schenk,Luke W. Guddat
出处
期刊:ACS Catalysis [American Chemical Society]
卷期号:14 (9): 6827-6841
标识
DOI:10.1021/acscatal.4c00958
摘要

NAD(P)H-dependent ketol-acid reductoisomerase (KARI) is the second enzyme in the branched-chain amino acid (BCAA) synthesis pathway and is also a vital (albeit rate-limiting) component of enzyme cascades to produce high-value chemicals such as isobutanol from renewable feedstocks. KARI catalyses two concerted reactions within a single active site, isomerization and reduction. Two distinct classes of KARI (I and II) have evolved to perform these complex reactions. Only Class II has been studied in detail, but Class I KARIs may be better suited for industrial applications in enzyme cascades due to the thermostability of some of its members. Here, all stages of the reaction cycle of the dodecameric Class I KARI from Camphylobacter jejuni (CjKARI) were visualized, and the mechanism probed by catalytic measurements to demonstrate that substrate binding needs to precede NADPH binding to initiate the reaction. NADPH binding exerts physical pressure on the substrate, promoting the isomerization reaction through controlled noncovalent interactions. The subsequent delocalization of one of the two Mg2+ ions in the active site then induces the reduction reaction. The extensive conformational changes and metal ion displacements that occur during the catalytic cycle explain the slow turnover rates experienced by this enzyme. However, the binding of K+ in a location remote from the active site leads to an ∼three-fold increase of the reaction rate, which has a significant impact on the commercial potential of the enzyme cascades in which KARI is integrated.
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