Crystal Structures of Staphylococcus aureus Ketol‐Acid Reductoisomerase in Complex with Two Transition State Analogues that Have Biocidal Activity

Abstract

Ketol-acid reductoisomerase (KARI) is an NAD(P)H and Mg -dependent enzyme of the branched-chain amino acid (BCAA) biosynthesis pathway. Here, the first crystal structures of Staphylococcus aureus (Sa) KARI in complex with two transition state analogues, cyclopropane-1,1-dicarboxylate (CPD) and N-isopropyloxalyl hydroxamate (IpOHA) are reported. These compounds bind competitively and in multi-dentate manner to KARI with K values of 2.73 μm and 7.9 nm, respectively; however, IpOHA binds slowly to the enzyme. Interestingly, intact IpOHA is present in only ≈25 % of binding sites, whereas its deoxygenated form is present in the remaining sites. This deoxy form of IpOHA binds rapidly to Sa KARI, but with much weaker affinity (K =21 μm). Thus, our data pinpoint the origin of the slow binding mechanism of IpOHA. Furthermore, we propose that CPD mimics the early stage of the catalytic reaction (preceding the reduction step), whereas IpOHA mimics the late stage (after the reduction took place). These structural insights will guide strategies to design potent and rapidly binding derivatives of these compounds for the development of novel biocides.

Mechanism and Inhibitor Exploration with Binuclear Mg Ketol‐Acid Reductoisomerase: Targeting the Biosynthetic Pathway of Branched‐Chain Amino Acids

“…Because the KARI‐catalyzed reaction was carried out at pH 8.0, the Lys130 residue was believed to be present in the protonated state whereas the Asp190, Glu194, and Glu226 residues were in the deprotonated states. Because the distance between one of Glu230 oxygen atoms and one of the CPD carboxyl oxygen atoms was observed to be very short (≈2.6 Å) in the crystal structure, which clearly indicates hydrogen bonding between them, both protonated and deprotonated Glu230 were calculated. Deprotonated Glu230 was found to give a barrier to hydride transfer 8.3 kcal mol −1 higher than that provided by protonated Glu230, thus making the reaction inaccessible (discussed in more detail in the section on mechanism A).…”

“…Biosynthesis of BCAAs exists only in bacteria, archaea, fungi, algae, and plants, the machinery for their generation being absent in mammals, so some biosynthetic pathways of BCAAs in plants and microorganisms might be exclusively suppressed, potentially without effects on mammals, by targeted inhibitors. Therefore, studies of the catalytic mechanisms in the biosynthesis of BCAAs could have significant impacts for the discovery of potent antifungal and antibacterial inhibitors and the exploitation of microorganisms for producing valuable chemicals and biofuels …”

“…Quite recently, the first X‐ray crystal structure of KARI from Staphylococcus aureus ( Sa KARI), in a complex with cyclopropane‐1,1‐dicarboxylate (CPD; PDB ID: 5W3K), was reported (Figure ). Its active site contains two divalent Mg 2+ cations, five coordinated water molecules, a NAD(P)H molecule, and the CPD inhibitor.…”

“… Overall view of KARI from S. aureus (in complex with CPD) and close‐up view of its active site. Coordinates from PDB ID: 5W3K were used to generate the pictures. In the right‐hand picture, Mg, C, O, and N atoms are shown in green, yellow, red, and blue, respectively.…”

“…The investigation and comparison of these two mechanisms might provide deeper insight into the KARI‐catalyzed reaction and potential inspiration for inhibitor exploration. Here we have investigated the reaction mechanisms of KARI with the aid of the density functional theory (DFT) method and a chemical model constructed on the basis of the above X‐ray crystal structure (PDB ID: 5W3K) . We present the energetics for the KARI‐catalyzed reaction and provide characterization of the stationary points involved.…”