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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.

Aminotransferases (ATs) are pyridoxal 5'-phosphate-dependent enzymes that catalyze the transamination reactions between amino acid donor and keto acid acceptor substrates. Modern AT enzymes constitute ∼2% of all classified enzymatic activities, play central roles in nitrogen metabolism, and generate multitude of primary and secondary metabolites. ATs likely diverged into four distinct AT classes before the appearance of the last universal common ancestor and further expanded to a large and diverse enzyme family. Although the AT family underwent an extensive functional specialization, many AT enzymes retained considerable substrate promiscuity and multifunctionality because of their inherent mechanistic, structural, and functional constraints. This review summarizes the evolutionary history, diverse metabolic roles, reaction mechanisms, and structure-function relationships of the AT family enzymes, with a special emphasis on their substrate promiscuity and multifunctionality. Comprehensive characterization of AT substrate specificity is still needed to reveal their true metabolic functions in interconnecting various branches of the nitrogen metabolic network in different organisms.

SUMMARY: Plants direct substantial amounts of carbon toward the biosynthesis of aromatic amino acids (AAAs), particularly phenylalanine to produce lignin and other phenylpropanoids. Yet, we have a limited understanding of how plants regulate AAA metabolism, partially because of a scarcity of robust analytical methods. Here, we established a simplified workflow for simultaneous quantification of AAAs and their pathway intermediates from plant tissues, based on extraction at two alternative pH and analysis by Zwitterionic hydrophilic interaction liquid chromatography coupled to mass spectrometry. This workflow was then used to analyze metabolic responses to elevated or reduced carbon flow through the shikimate pathway in plants. Increased flow upon expression of a feedback‐insensitive isoform of the first shikimate pathway enzyme elevated all AAAs and pathway intermediates, especially arogenate, the last common precursor within the post‐chorismate pathway of tyrosine and phenylalanine biosynthesis. Additional overexpression of an arogenate dehydrogenase enzyme increased tyrosine levels and depleted phenylalanine and arogenate pools; however, the upstream shikimate pathway intermediates remained accumulated at high levels. Glyphosate treatment, which restricts carbon flow through the shikimate pathway by inhibiting its penultimate step, led to a predictable accumulation of shikimate and other precursors upstream of its target enzyme but also caused an unexpected accumulation of downstream metabolites, including arogenate. These findings highlight that the shikimate pathway and the downstream post‐chorismate AAA pathways function as independently regulated modules in plants. The method developed here paves the way for a deeper understanding of the shikimate and AAA biosynthetic pathways in plants. Significance Statement: Aromatic amino acids (AAA) are key precursors to plant phenylpropanoids and other specialized metabolites. However, the regulation of AAA biosynthesis remains obscure partially due to the absence of robust methods for analyzing AAA pathway metabolites. Here, we report an extraction and LC–MS method for the simultaneous quantification of AAAs and their precursors from plant tissues. This methodology identified potentially regulated steps in the AAA pathways and will facilitate a deeper understanding of AAA metabolism. [ABSTRACT FROM AUTHOR]

SUMMARY: Plants produce a staggering array of chemicals that are the basis for organismal function and important human nutrients and medicines. However, it is poorly defined how these compounds evolved and are distributed across the plant kingdom, hindering a systematic view and understanding of plant chemical diversity. Recent advances in plant genome/transcriptome sequencing have provided a well‐defined molecular phylogeny of plants, on which the presence of diverse natural products can be mapped to systematically determine their phylogenetic distribution. Here, we built a proof‐of‐concept workflow where previously reported diverse tyrosine‐derived plant natural products were mapped onto the plant tree of life. Plant chemical‐species associations were mined from literature, filtered, evaluated through manual inspection of over 2500 scientific articles, and mapped onto the plant phylogeny. The resulting "phylochemical" map confirmed several highly lineage‐specific compound class distributions, such as betalain pigments and Amaryllidaceae alkaloids. The map also highlighted several lineages enriched in dopamine‐derived compounds, including the orders Caryophyllales, Liliales, and Fabales. Additionally, the application of large language models, using our manually curated data as a ground truth set, showed that post‐mining processing can largely be automated with a low false‐positive rate, critical for generating a reliable phylochemical map. Although a high false‐negative rate remains a challenge, our study demonstrates that combining text mining with language model‐based processing can generate broader phylochemical maps, which will serve as a valuable community resource to uncover key evolutionary events that underlie plant chemical diversity and enable system‐level views of nature's millions of years of chemical experimentation. Significance Statement: This study introduces a novel workflow to map the distribution of natural products onto a phylogeny. It also demonstrates the effectiveness of combining text mining of the literature with large language model evaluation for constructing a "phylochemical" database that can organize vast portions of the chemical diversity present across the plant tree of life. [ABSTRACT FROM AUTHOR]

Synthetic biology provides emerging tools to produce valuable compounds in plant hosts as sustainable chemical production platforms. However, little is known about how supply and utilization of precursors is coordinated at the interface of plant primary and specialized metabolism, limiting our ability to efficiently produce high levels of target specialized metabolites in plants. L-Tyrosine is an aromatic amino acid precursor of diverse plant natural products including betalain pigments, which are used as the major natural food red colorants and more recently a visual marker for plant transformation. Here, we studied the impact of enhanced L-tyrosine supply on the production of betalain pigments by expressing arogenate dehydrogenase (TyrA) from table beet (Beta vulgaris, BvTyrAα), which has relaxed feedback inhibition by L-tyrosine. Unexpectedly, betalain levels were reduced when BvTyrAα was coexpressed with the betalain pathway genes in Nicotiana benthamiana leaves; L-tyrosine and 3,4-dihydroxy-L-phenylalanine (L-DOPA) levels were drastically elevated but not efficiently converted to betalains. An additional expression of L-DOPA 4,5-dioxygenase (DODA), but not CYP76AD1 or cyclo-DOPA 5-O-glucosyltransferase, together with BvTyrAα and the betalain pathway, drastically enhanced betalain production, indicating that DODA is a major rate-limiting step of betalain biosynthesis in this system. Learning from this initial test and further debottlenecking the DODA step maximized betalain yield to an equivalent or higher level than that in table beet. Our data suggest that balancing between enhanced supply ("push") and effective utilization ("pull") of precursor by alleviating a bottleneck step is critical in successful plant synthetic biology to produce high levels of target compounds. Optimizing the push-and-pull strategy around tyrosine precursor boosts betalain production in Nicotiana benthamiana, opening avenues for efficient chemical production through plant synthetic biology. [ABSTRACT FROM AUTHOR]

The role of ferroptosis in steatohepatitis development is largely unknown. We investigated (1) whether hepatocyte ferroptosis occurs in a gene-modified steatohepatitis model without modifying dietary components, (2) whether ferroptosis occurs at an early stage of steatohepatitis, and (3) whether apomorphine, recently reported as a ferroptosis inhibitor, can ameliorate steatohepatitis. Hepatocyte-specific PTEN KO mice were used. Huh 7 and primary cultured hepatocytes isolated from the mice were used in this study. The number of dead cells increased in 10-week-old PTEN KO mice. This cell death was suppressed by the administration of ferroptosis inhibitor ferrostatin-1 for 2 weeks. Apomorphine also ameliorated the severity of steatohepatitis. Treatment with ferroptosis inhibitors, including apomorphine, decreases the level of lipid peroxidase. Apomorphine suppressed cell death induced by RSL-3 (a ferroptosis inducer), which was not suppressed by apoptosis or necroptosis inhibitors. Apomorphine showed a radical trapping capacity with much more potent activity than ferrostatin-1 and Trolox, a soluble form of vitamin E. In addition, apomorphine activated nrf2 and its downstream genes, including HO-1 and xCT. In conclusion, ferroptosis occurs in steatohepatitis from an early stage in PTEN KO mice. In addition, apomorphine ameliorates the severity of steatohepatitis by inhibiting ferroptosis. [ABSTRACT FROM AUTHOR]