Download Egri, Shawn March 23, 2015

Egri, Shawn
March 23, 2015
UVM Student Research Conference
Abstract Submission
Aminoacyl-tRNA synthetases (aaRSs) are a class of enzymes whose primary function is
aminoacylation, or the attachment of an amino acid to its corresponding tRNA. Along with
catalyzing this critical procedure many aaRSs have a secondary function as well. For ThreonyltRNA synthetase (ThrRS) this is promotion of angiogenesis, or blood vessel formation. Blood
vessel formation is problematic when it assists in metastasis; new vessels serve as superhighways
assisting in the spread of tumors from one area of the body to another. Inhibiting the secondary
function of ThrRS can be accomplished using the naturally-occurring inhibitor, borrelidin (BN).
Understanding the mechanism of BN inhibition has important pharmaceutical
implications. Previously, molecular modeling has suggested that BN operates by “locking”
ThrRS in its native conformation. Generating a mutant ThrRS construct which mimics the BNinhibited wild type enzyme can be useful in understanding the vitality of the conformational
change ThrRS undergoes during binding of canonical substrates. Studying the “locked”
conformation of ThrRS was completed by designing a mutant enzyme using PyMOL and sitedirected mutagenesis. The sequence of the mutant designed was verified by DNA analysis. This
mutant was explored via intrinsic tryptophan fluorescence, a technique that measures the
conformational change of ThrRS in the presence of canonical substrate, namely threonine (501000 μM). It was found that mutant ThrRS does not undergo the conformational change that
wild-type enzyme does, giving insight into the mechanism of BN action. By preventing a
conformational change, BN renders ThrRS inactive, preventing both its primary and secondary
functions. This indicates that the transformation of ThrRS upon canonical substrate binding is
vital to its functioning. In order to derive a pharmaceutical which has diminished toxicity relative
to BN and therefore has a broader clinical application, a new drug must allow the conformational
change of the enzyme, whilst simultaneously preventing its angiogenic activity.