Ketol-acid reductoisomerase

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Ketol-acid isomeroreductase (KARI) is the second important enzyme in the biosynthetic pathway of BCAAs. It can catalyze the production of 2-acetyl-2-hydroxybutyric acid to 2,3-dihydroxy-3-methylpentanoic acid to synthesize valine or isoleucine, and 2-acetyl-2-hydroxybutyric acid to 2,3-dihydroxy-3-methylpentanoic acid to synthesize leucine.

Gene

KARI is encoded by the ilv C gene and is divided into two types based on molecular weight, of which type I KARI consists of approximately 340 amino acid residues and is mainly found in some other bacteria such as Mycobacterium tuberculosis, Pseudomonas aeruginosa, and sugar beet nightshade. KARI exhibits a high degree of stereospecificity for the S-isomer of its substrate, and its catalytic reaction consists of two steps: first, an alkyl migration process, namely C2 The catalytic reaction consists of two steps: first, an alkyl migration process, in which the methyl or ethyl group at the C2 position migrates to the C3 position to produce an α-keto-β-hydroxy acid intermediate; then, a reduction process, using NAD(P)H to reduce the keto acid to the C2 hydroxyl product.

KARI protein structure and active site study

The active site of KARI is located at the interface of two structural domains, N and C, which bind two Mg²⁺ and NADPH as essential cofactors. n is composed of eight chains, each consisting of a β-fold with α-helices on either side to form a nucleotide-binding Rothman fold, C is composed of eight α-helices and forms a knotted structure. Analysis of the MtKARI crystal structure demonstrated that it exists as a dimer in solution and that this dimeric structure forms its active site. The active site is composed of the N-structure domain of one subunit of the dimer and the active site is formed by the N and C domains of one subunit of the dimer and the C domain of the other subunit, which is also observed in other type I KARI.

Properties

In addition to the involvement of NAD(P)H in the catalytic reaction of KARI, metal ions are also very important cofactors. It has been shown that KARI prefers Mg²⁺ as a cofactor and shows a strict Mg²⁺ dependence in the alkyl migration phase of its catalytic reaction. As one of the more important enzymes in the biosynthetic pathway of BCAAs, KARI has also been used as a target for screening effective antibacterial drugs and herbicides. IpOHA, a transition state analogue of KARI-catalyzed substrate to product conversion, has been studied not only as an herbicide but also exhibits some antimicrobial activity.

KARI inhibitor

Given the success of AHAS as an antimicrobial as well as herbicide target, other enzymes in the BCAA pathway may also have great potential. 2-dimethylphosphono-2-hydroxyacetic acid (Hoe704) and N-isopropyl oxalyl isohydroxamic acid (IpOHA), as transition state analogs in the conversion of KARI-catalyzed substrates to products, can effectively inhibit KARI activity and are potent and irreversible competitive inhibitor. The mechanism of inhibition is that the intermediate analogue binds to the active site of the enzyme, leading to the formation of an irreversible enzyme-inhibitor complex and inactivating the enzyme in a time-dependent manner.