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GatCAB: A Potential Target for Bacterial Destruction
Greenfield High School SMART Team: Morgan Borchardt, Srinidhi Emkay, Hannah Flees, Amanda Miller, Joey Krasovich, Robin Sandner,
Phat Nguyen, Panfua Thao, Tammy Tian, Tania Alvarez, Haleigh De Smet, Vivian Ramirez
Teacher: Julie Fangmann
Mentor: Martin St. Maurice, Ph.D., Marquette University
Abstract
Staph infection is caused by the bacteria Staphylococcus aureus, which have become
increasingly resistant to a broad spectrum of antibiotics. New ways to combat these
bacteria are needed. The Greenfield High School SMART (Students Modeling A Research
Topic) Team is modeling the enzyme GatCAB using 3D printing technology. GatCAB is found
in certain bacteria and archaea and could be a target for new antibiotics. During protein
synthesis, ribosomes bring together aminoacylated-tRNA molecules to form proteins
needed for survival. Some bacteria have tRNAs that always have an incorrect amino acid
attached. Staphylococcus aureus contains such misacylated tRNA molecules with aspartate
where asparagine should be attached. GatCAB’s three proteins (GatA, GatB, and GatC)
correct these misacylations. GatA’s active site produces ammonia, which travels through a
tunnel leading to GatB. GatC holds GatA and GatB together. GatB’s hinge recognizes and
binds to the T loop of the misacylated tRNA, so GatCAB will not correct a tRNA that should
have aspartate. The aspartate on the tRNA enters GatB’s active site, where the aspartate
reacts with ATP and the ammonia from GatA to form asparagine, ensuring the correct
amino acid is on the proper tRNA. If the misacylated tRNAs were left uncorrected, protein
synthesis would be severely disrupted, and the bacteria would die. If scientists produce a
drug to prevent GatCAB from fixing the misacylated amino acids, the world would have a
new weapon to fight antibiotic resistant bacteria.
MRSA and Antibiotic Resistance
•Staphylococcus aureus causes staph infection
•While antibiotics (such as penicllin and
amoxicillin,)can be used to kill bacteria, some
strains have become antibiotic resistant
•MRSA (Methicillin-Resistant Staphylococcus
aureus) is a strain resistant to a variety of
antibiotics
•Incidences of MRSA have risen over the years
(see graph)
•New ways to kill the bacteria are needed…
How GatCAB Fixes Asp-tRNA Misacylations
III. Ammonia Travels Through
the Tunnel
GatCAB with Asp-tRNA
GatA
GatA’s active site
The Tunnel
GatC
ATP in GatB’s active site
GatB
Misacylated aspartate
tRNA-Asp
tRNA’s T loop
GatB’s hinge
tRNA’s anticodon
One of the misacylations that occurs in some bacteria, such as Staphylococus aureus, occurs when an
aspartate (Asp) is placed where an asparagine (Asn) is supposed to be. The misacylated Asp-tRNA can be
fixed by GatCAB, as explained here.
Pdb file: GatCAB_tRNA.pdb
I. GatB hinge
recognizes and binds
to tRNA-Asp’s T-loop
II. Formation of Ammonia in
GatA’s Active Site
pdb file: GatCAB_tRNA.pdb
Protein Synthesis: The Basics
Protein synthesis is the process in which proteins are made. Proteins are composed
of chains of amino acids bonded together. Molecules called tRNA (transfer RNA) carry
single amino acids to a ribosome, where these amino acids get attached to one
another in a sequence based on the genetic information in the mRNA (messenger
RNA).
pdb file: GatCAB_tRNA.pdb
The T-loop (dark purple) on AsptRNA binds to the hinge (blue) on
GatB. The hinge will only bind to the
specific sequence of the T loop on
the misacylated tRNA. Once they
bind together, the aspartate (Asp)
inserts into the active site of GatB so
it can be fixed.
Ammonia is needed in order to change the misacylated
aspartate into the appropriate asparagine (Asn). The
active site in Gat A (light purple) is where the reaction
takes place. The reaction (below) uses water and serine165 (gray, blue and red above) in GatA to remove the
NH3+ from the asparagine. Once formed, the ammonia
will need to get to Gat B’s active site.
pdb file: GatCAB_tRNA.pdb
GatCAB has a tunnel (orange) between GatA and
GatB. The ammonia formed in GatA travels through
the tunnel to GatB’s active site, where it is needed.
IV. GatB’s Active Site Fixes the
Misacylated Asp-tRNA
pdb file: GatCAB_tRNA.pdb
The misacylated aspartate (magenta) on the tRNA is
already inserted into GatB’s active site, where ATP
(gray, blue, and red), ammonia (not present in
model), and the aspartate react to form asparagine.
[This reaction is essentially the reverse of that in
GatA’s active site (seen in box II.).] Finally, the
appropriate amino acid is on the Asn-tRNA.
V. Gat C Holds GatCAB
Together
pdb file: GatCAB_tRNA.pdb
Image supplied by Dr. Martin St. Maurice
Gat C (coral) works as a “belt” that keeps Gat A
and Gat B together. Without Gat C the whole
protein will fall apart and not function.
The Parts of tRNA That Interact with GatCAB
•The anticodon matches with a codon region on mRNA
to indicate which amino acid needs to be added.
•Each anticodon determines which specific amino acid
is supposed to be carried by the tRNA.
•A misacylated tRNA has an incorrect amino acid.
•Misacylations can cause proteins to malfunction,
possibly becoming detrimental to the organism.
•Each tRNA also has a unique T loop, which in this case
can assist in binding to GatCAB.
A SMART Team project supported by the National Institutes of Health Science Education Partnership Award (NIH-SEPA 1R25RR022749)
and an NIH CTSA Award (UL1RR031973).
GatCAB Could Be Used As a New Method for Killing Certain Types of Antibiotic Resistant Bacteria
As bacteria, such as Staphylococus aureus, become more resistant to a wide variety of
antibiotics, new ways to kill these disease-causing bacteria are needed. Blocking GatCAB’s
ability to fix misacylations might result in malfunctioning proteins and bacterial death.
Image A shows the
structures of Asp-tRNA
(when n= 1) and Glu-tRNA
(when n=2).
In a study conducted by Jonathon L. Huot, et. al., a competitive inhibitor of GatCAB
(aspartycin) was tested. Adding higher concentrations of aspartycin caused less activity by
GatCAB. Other similar inhibitors have also been tested, not all with the same outcomes.
Image B shows the
structure of the
competitive inhibitors,
aspartycin (when n=1) and
glutamycin (when n=2).
This figure shows that as the
concentrations of aspartycin [ ](or
glutamycin) [ ] increase, the
activity of GatCAB decreases. This
shows aspartycin and glutamycin
are competitive inhibitors of
GatCAB.
Images and data from: Huot, Jonathon L., et. al.; Mechanism of a GatCAB Amidotransferase: Aspartyl-tRNA Sythetase Increases Its Affinity for Asp-tRNAAsn and Novel Aminoacyl-tRNA Analogues Are Competitive Inhibitors; Biochemistry; 2007; 46; 13190-13198