New publication reveals broad substrate spectrum for a previously uncharacterized enzyme

Northen Lab researchers Markus de Raad, Benjamin Bowen, Kai Deng, and Trent Northen recently authored a research article in the Journal of Biological Chemistry investigating a new high-throughput method for screening aminotransferase (AT) activity. Although aminotransferases play a central role in nitrogen metabolism for all species, the functionality of these enzymes is not well understood due to a lack of high-throughput assays that can effectively screen for substrate utilization. In this study, we present a new high-throughput technology for characterizing aminotransferase activity and specificity using mass spectrometry-based enzyme assay, that overcomes challenges with limited throughputs and specificities of standard methods.

Nanostructure-initiator mass spectrometry (NIMS) is used, in combination with a perfluorous alkoxyamine probe that forms an oxime linkage with ketones that are present in any keto acid products after transamination of amino donors. The tagged products are printed onto the NIMS surface using acoustic sample deposition, and analyzed using mass spectrometry imaging (MSI).Using this new method, it was discovered that the previously uncharacterized Arabidopsis thaliana tryptophan AT-related protein 1 enzyme can utilize 13 amino acid donors and three keto acid acceptors. This work demonstrates that this new high-throughput method enables screening of AT amino donor and keto acceptor specificity, thus providing a better understanding of the nitrogen metabolic network.

To learn more, read the full article here.

Overview of the oxime-MSI AT assay. A, schematic overview of oxime tagging to detect AT activity. Tyrosine (Tyr) is, for example, transaminated by Tyr AT (TAT) using α-ketoglutarate (α-KG) as keto acceptor yielding 4-hydroxyphenylpyruvate (HPP) and glutamic acid (Glu). The aminooxy group on the oxime probe reacts with the ketone group of α-KG and HPP. The resulting covalent adduct is ready for subsequent MSI analysis. The amino donor involved in the AT reaction is shown in **red**, while the amine of the NIMS probe is in **blue**. B, steps in the oxime-MSI AT assay. After the enzymatic reactions and subsequent oxime tagging, samples are printed onto the NIMS surface using acoustic sample deposition. Next, MSI data is acquired and analyzed for AT activity, where positive signals are depicted as **green dots**. AT, aminotransferase; MSI, mass spectrometry imaging; NIMS, nanostructure-initiator mass spectrometry.

Reference:
de Raad, M., Koper, K., Deng, K., Bowen, B. P., Maeda, H. A., & Northen, T. R. (2023). Mass spectrometry imaging–based assays for aminotransferase activity reveal a broad substrate spectrum for a previously uncharacterized enzyme. Journal of Biological Chemistry, 299(3), 102939. https://doi.org/10.1016/j.jbc.2023.102939