Elucidation of the Protonation States of the Catalytic Residues in mtKasA: Implications for Inhibitor Design

Abstract

KasA (β-Ketoacyl ACP synthase I) is involved in the biosynthetic pathway of mycolic acids, an essential component of the cell wall in Mycobacterium tuberculosis. It was shown that KasA is essential for the survival of the pathogen and thus could serve as a new drug target to treat tuberculosis. The active site of KasA was previously characterized by X-ray crystallography. However, questions regarding the protonation state of specific amino acids, the orientation of the histidine groups within the active site, and additional conformers being accessible at ambient temperatures remain open and have to be addressed prior to the design of new inhibitors. We investigated the active site of KasA in the present work by means of structural motifs and relative energies. Molecular dynamics (MD) simulations, free energy perturbation (FEP) computations, and calculations employing the hybrid quantum mechanics/molecular mechanics (QM/MM) method made it possible to determine the protonation status and reveal important details about the catalytic mechanism of KasA. Additionally, we can rationalize the molecular basis for the acyl-transfer activity in the H311A mutant. Our data strongly suggest that inhibitors should be able to inhibit different protonation states because the enzyme can switch easily between a zwitterionic and neutral state.

Structural Basis for the Recognition of Mycolic Acid Precursors by KasA, a Condensing Enzyme and Drug Target from Mycobacterium Tuberculosis

“…Proposed KasA reaction mechanism. This mechanistic scheme is strongly supported by our structural work and builds on previous studies (13,44,54,55,57). Gray dashed lines indicate hydrogen bonds.…”

“…Based on our structural results concerning the binding mode of the malonyl group and taking previous findings into account (13,44,54,55), we propose the mechanism depicted in Fig. 8.…”

“…Consequently, we suggest that the initial product is CO 2 , which can then be hydrated after decarboxylation (between steps 4 and 5). The evolving enolate (box 5) is stabilized by the neutral His-345, which is protonated at its ⑀-nitrogen because the ␦-nitrogen is hydrogen-bonded to the backbone NH of Ile-347 (54). Geometric considerations based on our structures suggest that the nucleophilic attacks of Cys-171 and of the acetyl-carbanion occur from the Si-and Re-faces of the thioester carbonyl groups, respectively (boxes 2 and 5; see also Fig.…”

“…Positively (Lys-340; depicted in blue) and negatively (Asp-319, Glu-322, and Glu-354; depicted in red) charged amino acids, which were proposed to be important for decarboxylation (44), interact with His-311 via a complex hydrogen bonding network, which includes a potassium ion located in a conserved cation binding site (D). This environment probably leads to the modulation of the electronic properties of the His-311 ⑀-nitrogen (magenta) to a catalytically ideal state (54).…”

“…7E) and might thus be responsible for tailoring the catalytically ideal His-311 electronic state (54).…”