Mass spectrometry imaging-based assays for aminotransferase activity reveal a broad substrate spectrum for a previously uncharacterized enzyme.

eScholarship, University of California 2023-03-01

Aminotransferases (ATs) catalyze pyridoxal 5'-phosphate-dependent transamination reactions between amino donor and keto acceptor substrates and play central roles in nitrogen metabolism of all organisms. ATs are involved in the biosynthesis and degradation of both proteinogenic and nonproteinogenic amino acids and also carry out a wide variety of functions in photorespiration, detoxification, and secondary metabolism. Despite the importance of ATs, their functionality is poorly understood as only a small fraction of putative ATs, predicted from DNA sequences, are associated with experimental data. Even for characterized ATs, the full spectrum of substrate specificity, among many potential substrates, has not been explored in most cases. This is largely due to the lack of suitable high-throughput assays that can screen for AT activity and specificity at scale. Here we present a new high-throughput platform for screening AT activity using bioconjugate chemistry and mass spectrometry imaging-based analysis. Detection of AT reaction products is achieved by forming an oxime linkage between the ketone groups of transaminated amino donors and a probe molecule that facilitates mass spectrometry-based analysis using nanostructure-initiator mass spectrometry or MALDI-mass spectrometry. As a proof-of-principle, we applied the newly established method and found that a previously uncharacterized Arabidopsis thaliana tryptophan AT-related protein 1 is a highly promiscuous enzyme that can utilize 13 amino acid donors and three keto acid acceptors. These results demonstrate that this oxime-mass spectrometry imaging AT assay enables high-throughput discovery and comprehensive characterization of AT enzymes, leading to an accurate understanding of the nitrogen metabolic network.

Arabidopsis; Transaminases; Amino Acids; Spectrometry; Mass; Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity; Enzyme Assays; aminotransferases; enzyme promiscuity; enzyme screening; high-throughput screening; mass spectrometry; mass spectrometry imaging; nanostructure-initiator mass spectrometry; pyridoxal-5′-phosphate–dependent enzyme; Chemical Sciences; Biological Sciences; Medical and Health Sciences; Biochemistry & Molecular Biology; article

Open access content. Open access content
public

application/pdf
The Journal of biological chemistry vol 299, iss 3, 102939 0021-9258

CDLER oai:escholarship.org:ark:/13030/qt9w38s6hc
qt9w38s6hc
https://escholarship.org/uc/item/9w38s6hc
https://escholarship.org/
1391583350

UC MASS DIGITIZATION
From OAIster®, provided by the OCLC Cooperative.

edsoai.on1391583350