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Mycobacterium Delirium:
Inhibiting Leucine Biosynthesis to Starve M. tuberculosis
K Arnold, M Carrig, J David, J Diez, S Franczak, E Grogan, C Halloran, G Hilbert, S Michaeel, C Pfaff, M Pitts, C Rooney, T Rose, E Schauer, C Scherrer, T Siy, S Strandberg, J Strother
Teachers: S Strandberg, S Fleischmann
Mentor: Martin St. Maurice, PhD, Marquette University, Department of Biological Sciences
Introduction
The pathogen Mycobacterium tuberculosis represents a deadly threat to the worldwide population, especially poor and
developing countries, as it kills approximately 2 million people each year according to the World Health Organization.
Although it can affect any part of the body, M. tuberculosis most often infects the lungs and is transmitted in tiny droplets
released into the air by coughing and sneezing.
Figure 2. Healthy lungs
(left) vs. tuberculosisinfected lungs (right)
Figure 1. Symptoms,
transmission and
diagnosis of
tuberculosis
In recent years, the effectiveness of many antibiotics used to treat tuberculosis has decreased as multi drug-resistant strains
of tuberculosis have been identified in over 100 countries, so patients with active tuberculosis infections must take a myriad
of drugs to prevent the bacteria from developing further resistance. Because of overuse and increasing resistance to current
antibiotics, researchers are working to develop new drugs to more effectively treat tuberculosis.
Allosteric Regulation of
IPMS in Mycobacterium
New drugs that are not affected by existing bacterial
resistance mechanisms must be investigated. The repeated
use of the same drugs along with prolonged regimens
contributed to the emergence of strains that are resistant to
many of the drugs currently available to patients.
When leucine binds to the allosteric site in the regulatory
domain, it inhibits catalysis in the active site, shutting down
leucine biosynthesis. The leucine biosynthetic pathway is
necessary for the survival of Mycobacterium tuberculosis.
IPMS Molecular Structure
The IPMS protein structure is a dimer. Monomer A is highlighted khaki and monomer B is
highlighted blue.
The beta sheets are highlighted light blue.
Catalytic domain:
Active Site
- 5 Amino Acids (highlighted Fuchsia)
- Acetyl CoA
- Alpha KIV
- Zinc
Regulatory domain:
- Allosteric site for leucine is at
the subunit interface
- Will inhibit the enzyme and shut
down the leucine biosynthetic
pathway
Figure 3. Prevalence of multidrug
resistance in M. tuberculosis (darker
colors indicate greater prevalence)
Alpha-KIV is highlighted gold
When cellular leucine levels are low, the M. tuberculosis enzyme alphaisopropylmalate synthase (IPMS) catalyzes the condensation of acetylCoA with alpha-ketoisovalerate (alpha-KIV), the first step in the
production of the essential amino acid, leucine. As leucine levels
increase in the organism, leucine biosynthesis is shut down by its
binding to an allosteric site in the C-terminal domain of IPMS, which
inhibits the production of additional leucine. Researchers are working to
design a competitive inhibitor that would bind at the allosteric leucine
binding site and shut down the pathway, thus depriving M. tuberculosis
of this essential amino acid. The development of new drugs specific to
M. tuberculosis offers a promising way to overcome the problem of
antibiotic resistance and offers new tools to reduce life-threatening
tuberculosis infections.
TB Data
Prevalence of TB cases
Leucine is highlighted
pink
Potential Solution to
Mycobacterium Infection
Figure 4. Leucine biosynthetic
pathway in M. tuberculosis
Map of areas affected by TB
Zinc is highlighted
turquoise
TB cases by age and sex in the U.S.
Figure 5. Hydrogen – Deuterium exchange in one monomer of IPMS.
Regions highlighted in blue show decreased exchange in the presence of
leucine. Frantom et al (2009) Biochemistry 48, 7457.
Using a technique called Hydrogen – Deuterium exchange,
which follows a chemical reaction that replaces covalently
bonded hydrogen atoms with deuterium atoms, Frantom and
coworkers measured the flexibility of different regions of IPMS
in the presence and absence of leucine. H/D exchange
revealed that leucine binding rigidifies the regulatory domain
and part of the active site of IPMS. The rigidified IPMS is
unable to catalyze the reaction of acetyl-CoA with alpha-KIV,
preventing the biosynthesis of leucine.
The growing issue of antibiotic resistant bacteria has become increasingly difficult
to treat. Tuberculosis is one of the diseases that has been plagued by this growing
issue. Cutting off the essential leucine biosynthetic pathway offers a new possibility
to cure people who contract multidrug resistant tuberculosis. This strategy has the
potential to enhance or replace existing antibiotics targeting M. tuberculosis. By
inhibiting either the active site in the catalytic domain or activating the allosteric
site in the C-terminal domain, scientists would be able to shut off the biosynthetic
pathway for leucine; thus, preventing Mycobacterium tuberculosis from surviving in
the body.
The leucine biosynthetic pathway begins when IPMS catalyzes a reaction between
three ligands in its active site: alpha-KIV, acetyl-CoA, and Zn2+. Leucine will bind to
the enzyme to inhibit the the production of additional leucine, which can be taken
advantage of in drug development. Researchers would like to design a competitive
inhibitor that would interact at the leucine allosteric binding site which offers a
potent and specific way to shut down the pathway.
References
When a country is shaded
darker, there is a higher
incidence of TB in that location.
The nations with the highest
incidences of TB are often the
most underdeveloped.
This graph demonstrates that while the
number of TB cases among US-born people
(represented by blue bars) have decreased
consistently over the last fourteen years,
cases of TB among foreign-born persons
have remained fairly constant (represented
by gray bars).
This bar graph illustrates the incidence of
tuberculosis by age group and gender in
the U.S. As shown by the graph, the 65 +
age group has the highest incidence of TB.
The development of a new drug that inhibits the leucine
biosynthetic pathway may offer a new way to destroy
Mycobacterium tuberculosis and to cure the epidemic of drug
resistant tuberculosis. Additionally, leucine is an essential
amino acid, meaning that humans do not have a biosynthetic
pathway for leucine and must procure this essential amino
acid through their diet. Therefore, a drug that inhibits the
leucine biosynthetic pathway would only affect the infectious
bacteria and not the human host.
Koon, N., C. J. Squire, and E. N. Baker. "Crystal Structure of LeuA from Mycobacterium Tuberculosis, a Key Enzyme in Leucine
Biosynthesis." Proceedings of the National Academy of Sciences 101.22 (2004): 8295-300. Print.
"Antimicrobial Resistance." World Health Organization. N.p., Apr. 2015. Web. 8 Jan. 2016.
"Tuberculosis Trends — United States, 2014." Centers for Disease Control and Prevention. Centers for Disease Control
and Prevention, 20 Mar. 2015. Web. 8 Jan. 2016.
"2012 Surveillance Slides." Centers for Disease Control and Prevention. Centers for Disease Control and Prevention,
11 Sept. 2013. Web. 8 Jan. 2016.
"The SMART Team Program is supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Number 8UL1TR000055. Its
contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH."