Structural and mechanistic investigation towards the regulation of M.tb isocitrate lyase

Isocitrate lyase (ICL) is a crucial enzyme for the virulence and survival of Mycobacterium tuberculosis (M. tb) during infection. It is a key enzyme of the glyoxylate shunt and the methylcitrate cycle, metabolic pathways that are central to the bacteria’s carbon and lipid metabolism. The absence of ICL in mammals makes it a potential inhibition target for developing new treatments against tuberculosis. In M. tb, ICL exists in two isoforms, ICL1 and ICL2. Although numerous biochemical studies have been conducted to study M. tb ICL since the publication of the first ICL1 structure in 2000, key questions regarding the role of the two M. tb ICL isoforms and their modulation remain unresolved. The work described in this thesis investigates the regulation of ICL from structural and mechanistic perspectives. In particular, it focuses on the inhibition of M. tb ICL by the human macrophage metabolite itaconate, and the activation of M. tb ICL2 by lipid metabolite acetyl-coenzyme A (acetyl-CoA) and its structural analogues. The interactions between host and pathogen are important for M. tb as the bacteria may reside inside its human host for decades. Human macrophages produce itaconate as an antimicrobial metabolite. Previous studies have shown that it may act as an inhibitor of ICL, although its mode of action is not known. In this work, by using protein X-ray crystallography, mass spectrometry and mutagenesis, it was demonstrated that itaconate binds covalently to the active site cysteine of both M. tb ICL1 and ICL2. In addition, it was found that the presence of the ICL reaction product, glyoxylate, may enhance the rate of covalent modification. This work not only provides important structural information about the design of covalent ICL inhibitors, but it also provides insights into the catalytic mechanism of the enzyme, in particular the ‘back reaction’ that converts glyoxylate and succinate to itaconate. Hampered by the lack of structural information, the biological role of isoform 2 is not well understood. In this study, it was found that the activity of ICL2 can be activated through the binding of acyl-CoA (including acetyl-CoA and propionyl-CoA) to its C-terminal domain. As acyl-CoA is produced when fatty acids are used as a carbon source, this work showed that ICL2 is a gatekeeper in regulating carbon flow towards the glyoxylate shunt. Furthermore, by capitalising on the recently-solved M. tb ICL2 structures, studies were conducted to investigate how the binding of acyl-CoA may modulate the activity of the enzyme. ICL2 undergoes a striking global conformational change upon binding of acyl-CoA to its C-terminal domain, which involves the dimerisation of the C-terminal domains between neighbouring subunits (ICL2 is a tetrameric protein) and subsequent closure of the active site in the N-terminal catalytic domain. A series of biophysical assays were carried out to investigate how the binding of acyl-CoA may trigger the dimer rearrangement. Protein NMR spectroscopy and molecular dynamic simulations revealed that the C-terminal domain dimerisation is an equilibrium-driven process, with acyl-CoA stabilising towards the bound dimer state. It was found that the dimer-monomer dynamics in the presence of acyl-CoA match the catalytic rate, thus providing a rationale in how the C-terminal domain modulates the activity of ICL2. Overall, the work conducted in this thesis provides an in-depth analysis of the modulation of ICL by metabolites. It provides important information about how small molecules may be employed to inhibit the activity of ICL by targeting its catalytic cysteine through covalent interactions, as well as delivering unique mechanistic insights about the activation of M. tb ICL2. The most important finding of this work is that the binding of acyl-CoA to the C-terminal domain of ICL2 may activate ICL2, a process that is driven by changes in the position of the monomer-dimer equilibria of the ICL2 C-terminal domain. This discovery thus provides useful information in the future design of novel inhibitors and activators that target the regulatory domain of ICL2.

Name thesis:degree_name

PhD

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Chemistry

Grantor dc:publisher

ResearchSpace@Auckland

Year dc:date.issued

2022

Author dc:creator

• Kwai, Brooke X.C.

Advisors dc:contributor.advisor

• Leung, Ivanhoe
• Bashiri, Ghader

Statement dc:rights

• Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

Licence dc:rights.uri

• https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm

Handle dc:identifier.uri

https://hdl.handle.net/2292/61032

OAI identifier oai:identifier

oai:researchspace.auckland.ac.nz:2292/61032