Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
Department of Botany, University of Wisconsin-Madison; Madison, Wisconsin, USA.
J Biol Chem. 2023 Mar;299(3):102939. doi: 10.1016/j.jbc.2023.102939. Epub 2023 Jan 24.
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.
氨基转移酶（ATs）催化吡哆醛 5'-磷酸依赖的氨基供体和酮受体底物之间的转氨基反应，在所有生物的氮代谢中发挥核心作用。ATs 参与蛋白源和非蛋白源氨基酸的生物合成和降解，并且在光呼吸、解毒和次生代谢中也执行广泛的各种功能。尽管 ATs 非常重要，但它们的功能知之甚少，因为只有一小部分来自 DNA 序列预测的假定 ATs 与实验数据有关。即使对于特征明确的 ATs，在大多数情况下，在许多潜在底物中，其底物特异性的全部范围也尚未得到探索。这在很大程度上是由于缺乏合适的高通量测定法，这些测定法可以在大规模上筛选 AT 活性和特异性。在这里，我们提出了一种使用生物共轭化学和基于质谱成像的分析筛选 AT 活性的新高通量平台。通过在被转氨基的氨基供体的酮基与促进基于质谱的分析的探针分子之间形成肟键来检测 AT 反应产物，该探针分子使用纳米结构引发剂质谱或 MALDI 质谱进行分析。作为原理验证，我们应用了新建立的方法，发现先前未表征的拟南芥色氨酸 AT 相关蛋白 1 是一种高度混杂的酶，可利用 13 种氨基酸供体和 3 种酮酸受体。这些结果表明，这种肟-质谱成像 AT 测定法能够实现 AT 酶的高通量发现和全面表征，从而准确理解氮代谢网络。
