Researcher(s)
- Anoushka Buddhikot, Chemical Engineering, University of Delaware
Faculty Mentor(s)
- Aditya Kunjapur, Chemical and Biomolecular Engineering, University of Delaware
Abstract
ꞵ-hydroxy ɑ-amino non-standard amino acids (ꞵ-OH nsAAs) is a category of organic molecules that have manifold biological applications. For instance, Droxidopa is a ꞵ-OH nsAA that can be used to treat orthostatic hypotension (low blood pressure caused by standing) symptomatic of Parkinson’s disease1. Additionally, antibiotics such as chloramphenicol and vancomycin can be chemically derived from ꞵ-OH nsAAs4. L-Threonine transaldolases (TTAs) are a class of enzymes that catalyze the conversion of aromatic aldehydes and L-threonine to ꞵ-OH nsAAs and acetaldehyde. Enzymatic synthesis of these molecules is especially valuable because biosynthesis results in increased yield at a lower cost and environmental impact than traditional chemical synthesis. Additionally, TTAs are enantioselective for L-isomers of ꞵ-OH nsAA products and are associated with high diastereomeric excess of the L-threo diastereomer. This research examines the reaction catalyzed by TTAs and aims to improve yield of the ꞵ-OH nsAA by optimizing media conditions for in vitro TTA activity. Specifically, the effects of pH, buffer composition, cofactor concentration and substrate concentration on enzyme productivity were investigated using in vitro 96-well plate assays that varied each parameter. The reactions were incubated at 30°C and were quenched using trifluoroacetic acid. The contents of each well following the reaction were analyzed by high performance liquid chromatography (HPLC) to determine the depletion of aromatic aldehyde and the production of ꞵ-hydroxy amino acid.
Preliminary results demonstrate that the TTA enzyme has increased productivity under basic pH conditions and we observed product formation across a range of L-threonine concentrations. Additionally, supplemental titers of the cofactor pyridoxal 5’ phosphate (PLP) do not appear to affect product formation, potentially due to PLP that is bound to the TTAs during overexpression and purification. Our future research will continue to examine the effects of pH on TTA productivity and whether similar results hold for alternate aromatic aldehyde substrates. This optimization will pave the way for future engineering of in vitro multi-enzyme cascades for the biosynthesis of various ꞵ-OH nsAAs and their derivatives.
1. Kaufmann H, Norcliffe-Kaufmann L, Palma JA. Droxidopa in neurogenic orthostatic hypotension. Expert Rev Cardiovasc Ther. 2015;13(8):875-891. doi:10.1586/14779072.2015.1057504.
2. Scott, T. A.; Heine, D.; Qin, Z.; Wilkinson, B. An L-Threonine Transaldolase Is Required for L-Threo-β-Hydroxy-α-Amino Acid Assembly during Obafluorin Biosynthesis. Nature Communications 2017, 8 (1). DOI:10.1038/ncomms15935.