Probing the Catalytic Mechanism Involved in the Isocitrate Lyase Superfamily: Hybrid Quantum Mechanical/Molecular Mechanical Calculations on 2,3-Dimethylmalate Lyase

Abstract

The isocitrate lyase (ICL) superfamily catalyzes the cleavage of the C(2)-C(3) bond of various α-hydroxy acid substrates. Members of the family are found in bacteria, fungi, and plants and include ICL itself, oxaloacetate hydrolase (OAH), 2-methylisocitrate lyase (MICL), and (2R,3S)-dimethylmalate lyase (DMML) among others. ICL and related targets have been the focus of recent studies to treat bacterial and fungal infections, including tuberculosis. The catalytic process by which this family achieves C(2)-C(3) bond breaking is still not clear. Extensive structural studies have been performed on this family, leading to a number of plausible proposals for the catalytic mechanism. In this paper, we have applied quantum mechanical/molecular mechanical (QM/MM) methods to the most recently reported family member, DMML, to assess whether any of the mechanistic proposals offers a clear energetic advantage over the others. Our results suggest that Arg161 is the general base in the reaction and Cys124 is the general acid, giving rise to a rate-determining barrier of approximately 10 kcal/mol.

Demystifying the catalytic pathway of Mycobacterium tuberculosis isocitrate lyase

“…The figure showed that a concerted catalytic mechanism is unlikely since the deprotonation of isocitrate C(2)–OH and a proton transfer to the acceptor residue occurs before the elongation of the C(2)–C(3) bond and thereafter leading to bond breaking. This is consistent with the observation for the mechanism of DMML, a subclass of the lyase family 16 . The starting structure for the non-constrained transition state was obtained from the scan coordinate (Fig.…”

“…In DMML, Arg161 (corresponding to Arg228 of M. tuberculosis ICL) was reported to be potentially responsible for the deprotonation of isocitrate C(2)–OH which leads to the cleavage of isocitrate C(2)–C(3) bond 16 , 20 . However, our study showed alternative findings as ICL Arg228 is already in the protonated state and could not serve as a proton shuttle from isocitrate C(2)–OH that leads to the isocitrate cleavage.…”

“…Density functional theory (DFT) was applied in for QM optimization. M06-2X functional 16 , 55 in conjunction with 6-31+G(d,p) basis set was used to obtain minima and transition states optimized geometries. The electrostatic interaction between the QM and MM region was described by the electronic embedding scheme 51 , 56 implemented in ONIOM.…”

“…The second proposed mechanism involved Tyr43 as a catalytic base in the transfer of proton to His114 bonded to water molecules, resulting in the shuttling of a proton from isocitrate C(2)–OH group to Glu115 17 , 18 . Besides, Arg161 and Tyr89 of MICL (Arg 228 and Tyr43 in ICL) were also proposed as the unidentified catalytic base 16 , 20 .…”

“…The hybrid quantum mechanics/molecular mechanics (QM/MM) approach has been used to gain insight into the reaction mechanism of enzymes 21 – 23 . The QM/MM approach has also been applied to understand the catalytic mechanisms for lyases such as aspartate ammonia lyase 24 , N-acetylneuraminic acid lyase 25 , α-1,4-glucan lyase 26 , ( R )-hydroxynitrile lyase 27 and 2,3-dimethylmalate lyase (DMML) 16 . Studies on IMP dehydrogenase, pectate/pectin lyases, fumarate reductase, and l -aspartate oxidase 28 , as well as citrate synthase 29 suggested that Arg could be the catalytic base while calculations on DMML 16 and pyruvate formate-lyase 30 proposed that Cys as the catalytic acid.…”