Mechanisms Of Mycolic Acid Generation In Mycobacteria
Lead Research Organisation:
University of Birmingham
Department Name: Sch of Biosciences
Abstract
Tuberculosis (TB) is a bacterial infectious disease affecting humans and cattle. The human form of tuberculosis is caused by Mycobacterium tuberculosis, and has been a global health concerns due to the high global mortality rates and the rise and spread of difficult-to-treat, strains of the causative agent that are resistant to most drugs used for treating TB. The bovine version of TB, caused by Mycobacterium bovis, also has a significant impact on local agriculture costing the UK taxpayer over £100 million each year.
The cell envelope of the TB bacillus is a unique and 'waxy' layer made up of unique lipids (fats). One class of such lipids, termed mycolic acids, are a critical component of the cell envelope. Mycolic acids play an important role in the ability of the pathogen to cause disease, however they are also an Achilles heel of the bacterium. Mycobacteria cannot survive without mycolic acids and thus the genes involved in making mycolic acids are essential to keep the bacteria alive. This gives us an opportunity to target mycolic acid biosynthesis genes for future drug development.
In this study, we aim to understand the final, finishing stages of how mycolic acids are made, with a focus of two enzymes involved in the finishing steps, prior to transport and deposition on the outside of the cell. The first enzyme, polyketide synthase 13 (Pks13) brings together two large units and condenses them into a precursor for the finished product. Here, our preliminary work suggest new, previously unidentified sections of Pks13 that potentially form a new domain that drives two molecules of Pks13 to form a dimer, proposing new mechanistic insights into how this enzyme works. Our main approach involved purifying the protein and individual domains and use a technique called cryo electron microscopy to demonstrate the existence of these structures by validating our predicted models for how these domains of Pks13 look like in 3D. We will also probe the potential interaction of Pks13, with the another enzyme we aim to study in this proposal: the gene for MmrA has been shown to be essential for the 'completion' of mycolic acid biosynthesis. We will probe the potential interaction of Pks13 with MmrA, using purified proteins as well as genetic systems that enable the testing of protein-protein interactions.
The mechanisms of how MmrA functions are also not clear, and in this study we aim to shed light on this process by determining the substrate for MmrA, by testing an assay for the purified enzyme that will help us measure its activity. Overall, our studies will outline the final stages of mycolic acid production, and highlight the potential of these steps to be targeted as unique targets for drug development in the future.
The cell envelope of the TB bacillus is a unique and 'waxy' layer made up of unique lipids (fats). One class of such lipids, termed mycolic acids, are a critical component of the cell envelope. Mycolic acids play an important role in the ability of the pathogen to cause disease, however they are also an Achilles heel of the bacterium. Mycobacteria cannot survive without mycolic acids and thus the genes involved in making mycolic acids are essential to keep the bacteria alive. This gives us an opportunity to target mycolic acid biosynthesis genes for future drug development.
In this study, we aim to understand the final, finishing stages of how mycolic acids are made, with a focus of two enzymes involved in the finishing steps, prior to transport and deposition on the outside of the cell. The first enzyme, polyketide synthase 13 (Pks13) brings together two large units and condenses them into a precursor for the finished product. Here, our preliminary work suggest new, previously unidentified sections of Pks13 that potentially form a new domain that drives two molecules of Pks13 to form a dimer, proposing new mechanistic insights into how this enzyme works. Our main approach involved purifying the protein and individual domains and use a technique called cryo electron microscopy to demonstrate the existence of these structures by validating our predicted models for how these domains of Pks13 look like in 3D. We will also probe the potential interaction of Pks13, with the another enzyme we aim to study in this proposal: the gene for MmrA has been shown to be essential for the 'completion' of mycolic acid biosynthesis. We will probe the potential interaction of Pks13 with MmrA, using purified proteins as well as genetic systems that enable the testing of protein-protein interactions.
The mechanisms of how MmrA functions are also not clear, and in this study we aim to shed light on this process by determining the substrate for MmrA, by testing an assay for the purified enzyme that will help us measure its activity. Overall, our studies will outline the final stages of mycolic acid production, and highlight the potential of these steps to be targeted as unique targets for drug development in the future.
Technical Summary
The cell wall of the causative agent of the human (and bovine) disease tuberculosis (TB) is distinct from other bacterial cell envelopes due to a lipid-rich outer layer. Major components of this waxy envelope are long chain alpha-alkyl, beta-hydroxy fatty acids called mycolic acids that are essential for viability and virulence. While mycolic acid biosynthesis and transport mechanisms are well studied, we currently don't fully understand the post biosynthesis processing of mycolic acids, that intersperses these two processes. In this proposal, we aim to outline the molecular mechanisms that drive this late-stage biosynthesis by studying two key enzymes involved in this process:
1. Pks13, a polyketide synthase that catalyses the Claisen condensation of the two components , mero chain and alpha branch to produce alpha-alkyl, beta-keto precursors of mycolic acids.
2. MmrA, a reductase that is responsible for converting the product of Pks13 into mycolic acids via the reduction of the beta-keto group to a hydroxy.
This knowledge will inform therapeutic strategies directed at inhibiting mycolic acid synthesis, and shed light on a unique mycobacterial synthetic pathway. We will achieve this aim by:
1 Determining the structure of a newly indentified triple domain unit ACP2-pseudo ACP-TE (Pks13 residues 1225-1733) using X-ray crystallography and/or cryoEM.
2 Characterising the overall assembly and conformational state(s) of Pks13, including the triple-domain unit using a combination of cryoEM, SAXS and mutagenesis
3 Validating the newly identified structural features/domains of Pks13 in vivo using a Corynebacterium glutamicum pks13 mutant complemented with plasmid encoded M. tuberculosis pks13 mutant alleles.
4 Verify the role of trehalose mono keto-mycolate as the substrate for mycolyl reductase MmrA.
5 Validating co-folding models testing the interaction of Pks13 with MmrA using cryoEM, and protein-protein interaction and mutagenesis studies.
1. Pks13, a polyketide synthase that catalyses the Claisen condensation of the two components , mero chain and alpha branch to produce alpha-alkyl, beta-keto precursors of mycolic acids.
2. MmrA, a reductase that is responsible for converting the product of Pks13 into mycolic acids via the reduction of the beta-keto group to a hydroxy.
This knowledge will inform therapeutic strategies directed at inhibiting mycolic acid synthesis, and shed light on a unique mycobacterial synthetic pathway. We will achieve this aim by:
1 Determining the structure of a newly indentified triple domain unit ACP2-pseudo ACP-TE (Pks13 residues 1225-1733) using X-ray crystallography and/or cryoEM.
2 Characterising the overall assembly and conformational state(s) of Pks13, including the triple-domain unit using a combination of cryoEM, SAXS and mutagenesis
3 Validating the newly identified structural features/domains of Pks13 in vivo using a Corynebacterium glutamicum pks13 mutant complemented with plasmid encoded M. tuberculosis pks13 mutant alleles.
4 Verify the role of trehalose mono keto-mycolate as the substrate for mycolyl reductase MmrA.
5 Validating co-folding models testing the interaction of Pks13 with MmrA using cryoEM, and protein-protein interaction and mutagenesis studies.