Enzymes are nature’s wizards: balanced delicately on the margin of order and entropy, they perform chemical reactions and syntheses at rates and yields human chemists can only dream of. Many possess exquisite control mechanisms to keep the flow of metabolites through our cells precisely regulated. This work explores the regulation mechanism of α-isopropylmalate synthase (α-IPMS).
The branched-chain amino acid biosynthetic pathways in bacteria are of interest as novel antibiotic targets. α-IPMS catalyses the first committed step in the pathway to form leucine, an essential amino acid. It performs the Claisen condensation of α-ketoisovalerate (α-KIV) and acetyl coenzyme A (AcCoA) to form α-isopropylmalate (α-IPM). Almost all previously characterised α-IPMS enzymes are feedback regulated by leucine, the end-product of this pathway.
This study uses the α-IPMS enzymes from two pathogenic species, Myco- bacterium tuberculosis and Neisseria meningitidis (MtuIPMS and NmeIPMS, respectively). These enzymes are homodimeric in solution, and have a catalytic dimer of (β/α)8 barrels. This is connected via two more subdomains to a dimerised C-terminal regulatory domain, where leucine binds. The crystal structures of MtuIPMS with and without leucine bound are almost identical. Thus, we do not yet fully understand the mechanisms by which leucine is
recognised, nor how the allosteric signal is conducted ̴ 50 Å from the regulatory domain to the active site, and how this disrupts catalysis.
Chapter 2 explores the residues responsible for recognising and binding leucine. We use insights from the partial crystal structure of a similar enzyme in Leptospira interrogans, citramalate synthase (CMS). CMS catalyses a similar reaction to α-IPMS: the condensation of AcCoA and α-ketobutyrate (α-KB) to form citramalate, as the first step in isoleucine production in this organism. CMS is feedback regulated by isoleucine just as α-IPMS is regulated by leucine. CMS also shares a very similar overall structure to α-IPMS, and four conserved residues in each enzyme were identified as being responsible for binding the allosteric effector. In previous work, Tyler Clarke1 mutated each of the four MtuIPMS residues to the corresponding residue from LiCMS in an attempt to make an isoleucine-regulated MtuIPMS. While one mutant did show an increased sensitivity to the related amino acid norvaline, none of these mutations by themselves were sufficient to create an isoleucine-sensitive MtuIPMS. This work found that by using certain combinations of these mutations, we were able to create isoleucine-inhibited α-IPMS enzymes.
Dr. Wanting Jiao has been using molecular dynamics simulations to identify the residues important for allosteric signal propagation and disrupting catalysis in NmeIPMS . Chapter 3 details several of these residues which we have mutated, and presents the preliminary results of activity and inhibition studies on the mutant enzymes.
Chapter 4 summarises our findings and outlines the work required to further our understanding of the allosteric control systems studied here. Adapting the power of enzymes to contribute to the development of green chemistry, biosensors, and new antibiotics may prove to be one of the greatest opportunities ahead of modern chemistry.
| Identifer | oai:union.ndltd.org:canterbury.ac.nz/oai:ir.canterbury.ac.nz:10092/10849 |
| Date | January 2015 |
| Creators | Davies, Andrew |
| Publisher | University of Canterbury. Department of Chemistry |
| Source Sets | University of Canterbury |
| Language | English |
| Detected Language | English |
| Type | Electronic thesis or dissertation, Text |
| Rights | Copyright Andrew Davies, http://library.canterbury.ac.nz/thesis/etheses_copyright.shtml |
| Relation | NZCU |
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