Progress 07/01/24 to 06/30/25
Outputs
Target Audience:The target audience of this multidisciplinary project is broad, from basic plant biochemists to agriculture industry that are interested in improving crop nutritional values as well as pharmaceutical industry that is interested in improving the production of plant natural products. Toward this target audience in mind, we started biochemical experiments as well as genetic analyses in model plants, such as Arabidopsis and Nicotiana, but then we will also carry out studies in crops, particularly soybean. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Maeda lab: Besides senior graduate student, Soyoung Jung, a new graduate student, Carly Sanders, and a new postdoc, Charlay Wood, joined the project in December 2024 and conducted transient expression of variou PAL and other related enzymes in Nicotiana benthamiana leaves. They were trained in a number of experimental procedures, including the Golden Gate cloning, Agrobacterium-mediated plant transformation, and LC-MS/MS based metabolite profiling. Jez lab: Jae Morris (postdoc) and Daniel Berkovich (post-baccalaureate research technician) have worked on structural studies outlined in aim 1. Both were trained on experimental protocols, including protein expression, purification, and assays related to the project, as well as structural biology methods from protein crystallization to data collection to structure determination and interpretation. In addition, three graduate students (Wynne Havranek, Nushrat Rashid, Jesus Peng Zhao) worked with the PAL/PTAL proteins from this project during their first-year rotations to learn basic biochemical techniques described above. How have the results been disseminated to communities of interest?Members from the Maeda labcarried out in-person outreach events to disseminate our work to the general public and the importance of plant-derived metabolites to human well-being and society. During the UW Science Expedition, we conducted "Pigment Art" activity with K-12 children, who carried out paper chromatography/chemical reactions with plant pigments to learn about their properties and their importance in our society. We also carried out a Science Night community outreach event by visiting Heugel Elementary School in Madison, WI. In addition to the pigment activity, we also used a "metabolic pathway" jigsaw puzzle for children to learn how different pigments are made from CO2 and sunlight energy through plant metabolic pathways. Jez lab: The project was highlighted in a presentation by Dr. Jez to the Washington University Elliot Society's "Food & Agriculture Research Mission Community Event." This project and related work from the Maeda & Jez labs were also covered in ASBMB Today (the American Society of Biochemistry and Molecular Biology's monthly magazine) article on "From lab to land: modifying crops to tackle tomorrow's climate." What do you plan to do during the next reporting period to accomplish the goals?In the following reporting period, we will complete and submit the first manuscript to report the discovery of atypical non-canonical PAL enzymes, which has already been filed of U.S. patent. We will continue to conduct detailed biochemical characterization and structure analyses of atypical non-canonical PAL enzymes from different plant groups. We will be also testing the impacts of expressing PTAL and deregulated PTAL to investigate how the dual entry pathways will impact phenylpropanoid biosynthesis. We will be transforming soybean plants with deregulated DHS and PAL/PTAL enzyme coding genes to test in crops how deregulating the two critical steps will impact the overall plant metabolism, in particular aromatic chemical production. During the next year, Jez lab will focus on biochemical, mutagenesis, and structural analyses of EgPALnc and the newly identified ligand binding site to evaluate its impact on enzyme function and regulation. For example, additional co-crystallization and ligand binding experiments with aromatic ligands will be performed and the impact of site-directed mutants in the site examined. We also will continue protein expression, purification, and crystallization efforts of other PAL/PTAL proteins.
Impacts
What was accomplished under these goals?
Aim 1 Determine the molecular basis of PAL regulation, through phylogeny-guided sequence-structure-function analyses. To understand the molecular basis of phenylalanine ammonia lyase (PAL) regulation by phenylpropanoids, we have produced in recombinant form and purified 20 PALs (both canonical and non-canonical) and phenylalanine/tyrosine ammonia lyases (PTALs), and tested their sensitivity to feedback inhibition by various phenylpropanoids and products of Phe and Tyr catabolism. These PALs and PTAL genes were selected using a phylogeny-guided approach to represent the diversity of PAL isoforms from bryophytes to flowering plants, with a particular emphasis in selecting previously uncharacterized PALs, including non-canonical PALs from flowering plants. The canonical PAL enzymes were predominantly very sensitive to inhibition by trans-cinnamic acid, generally reaching 75 to 95% inhibition. Phenylpyruvate, product of Phe transamination, was also a strong inhibitor of canonical PALs (75-95% inhibition). However, the other compounds had relatively little inhibitory effect on PALs. Conversely, PTAL enzymes were very sensitive (>80% inhibition) to inhibition by all compounds tested except ferulic acid (only ~50% inhibition) when assayed with Phe to measure their PAL activity. When assayed with Tyr for TAL activity, PTALs were overall less sensitive to this feedback inhibition, although p-coumaric acid and 4-hydroxyphenylpyruvate were still very strong inhibitors (>90% inhibition), with moderate inhibition (~50%) caused by caffeic acid, phenylpyruvate and homogentisate. Among the non-canonical PALs tested, cotton GrPALnc and kava PmPALnc1 showed similar inhibition profiles to those of canonical PALs. In contrast, the non-canonical PALs from Elaeis guineensis (oil palm) EgPALnc and kava PmPALnc2 not only showed notable resistance (only 30~50% inhibition) against inhibition by trans-cinnamic acid and phenylpyruvate, but also a unique +50% activation by caffeic acid, p-coumaric acid and 4-hydroxyphenylpyruvate. To obtain a more accurate quantification of PAL and PTAL sensitivity to feedback inhibition, we measured the semi-inhibitory concentration (IC50) of trans-cinnamic acid across these 20 PAL/PTALs. Notably, within the group of highly resistant enzymes, EgPALnc and PmPALnc2 showed the highest resistance to tCA, with IC50 beyond 1.3 mM. When we quantified the activity of EgPALnc and PmPALnc2, as well as the feedback-inhibited AtPAL1 as a control, at different concentrations of caffeic acid, AtPAL1 was inhibited by caffeic acid with an IC50 of ~500 μM, whereas EgPALnc and PmPALnc2 were activated by +60% at this same concentration. These findings show that different PAL and PTAL enzymes exhibit diverse sensitivity to feedback regulation by phenylpropanoids and other Phe and Tyr derived metabolites. Notably, the non-canonical enzymes EgPALnc and PmPALnc2 have reduced sensitivity to inhibition and even are activated by certain phenylpropanoids. During year 1 of this project, the Jez lab has made substantial progress on structural studies of canonical and non-canonical PAL/PTALs. We have successfully determined the first x-ray crystal structure of a non-canonical PAL from palm EgPALnc. This three-dimensional structure (2.35-Å resolution) confirms the shared overall topology of the canonical and non-canonical PALs. Interesting, the structure also identified a ligand binding site along the oligomer interface between each monomer in the tetramer. This suggests a possible site to explore as the basis for the regulatory differences between the canonical and non-canonical forms of PAL. We also determined the x-ray crystal structures of wild-type Joinvillea ascendens PAL (JaPAL) in apoenzyme (2.9-Å resolution) and tyrosine bound (2.75-Å resolution) forms. These structures provide insight on the conformational changes associated with a mobile active site loop, which includes a critical catalytic tyrosine. Binding of the weak substrate tyrosine to JaPAL suggests how additional structural changes are required for optimal catalysis, which were revealed by structural studies of a JaPAL mutant enzyme engineered to have PTAL activity. Biochemical experiments, along with bioinformatic analysis, revealed that the evolution of PTAL function in plants required multiple active site changes, not just a single point mutation as observed for bacterial PTALs (Watts et al., 2006). Studies of JaPAL demonstrate that introduction of a histidine (Phe in PAL; His in PTAL) in the substrate binding site and a serine (Ile in PAL; Ser in PTAL) on the mobile active site loop are required for conversion of activity. The x-ray crystal structures of the JaPAL I112S/F140H mutant in apoenzyme (2.95-Å resolution) and tyrosine bound (2.8-Å resolution) forms identify how these changes alter positioning of the substrate tyrosine in the active site. In addition, direct experimental evidence (i.e., electron density) of the adduct formed between the substrate tyrosine amine group and the active site prosthetic group MIO (3,5-dihydro-5-methyldiene-4H-imidazol-4-one) conclusively resolves the chemical reaction mechanism of this enzyme, as a Friedel-Crafts reaction and not an E1/E2-elimination. Aim 2: Examine in planta functions of various PAL enzymes having altered regulation. To test in planta functions of various PALs, we transiently expressed canonical and non-canonical PAL/PTALs in the leaves of Nicotiana benthamiana. We compared the effects of expressing the feedback-insensitive non-canonical PALs EgPALnc and PmPALnc2, using the feedback-inhibited canonical AtPAL1 as control. These PALs were co-infiltrated with a gene construct carrying a feedback-insensitive DHS1sotaB4 mutant for enhancing Phe production in planta. LC-MS analysis of the infiltrated plant tissue at four days post-infiltration revealed that plants expressing EgPALnc and PmPALnc2 accumulated around 20-times higher levels of a putative trans-cinnamic acid glycoside ester, around 12-times higher levels of p-coumaric acid, and around 3-times higher levels of ferulic acid, compared to plants expressing AtPAL1. The plants expressing EgPALnc and PmPALnc2, compared to AtPAL1 expressing plants, also had lower levels of phenylalanine, tyrosine, 4-hydroxyphenylpyruvate and phenylpyruvate, which are otherwise very highly accumulated when DHS1sotaB4 is expressed. Detection of EgPALnc, PmPALnc2 and AtPAL1 in the plant samples by Western blot revealed that EgPALnc and PmPALnc2 proteins were much more abundant, around 10-times, than AtPAL1, suggesting that increased stability of non-canonical PALs also contributes to their high activity in planta. To further evaluate the in planta functionality of non-canonical PALs compared to other previously characterized PALs, we have made plant expression constructs for stable transformation of EgPALnc and AtPAL1 (as control) in Arabidopsis thaliana. These constructs have been transformed into wild-type Col-0 plants as well as sotaB4 mutant plants having hyper-accumulation of all AAAs, most substantially Phe, due to the deregulation of the upstream shikimate pathway (Yokoyama et al. 2022 Sci. Adv 8, eabo3416). We are currently isolating homozygous lines for the transgenic lines in Col-0 and sotaB4 Arabidopsis backgrounds. Once isolated, these homozygous transgenic plants will be analyzed by LC-MS to determine the levels of phenylpropanoids by targeted and untargeted analysis, as previously done for transient expression experiments in Nicotiana benthamiana. The discovery of these atypical PALs that effectively metabolize phenylalanine and dramatically enhance phenylpropanoid intermediates has been filed for U.S. provisional patent in May 2025 and is currently in preparation for publication.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
Atypical phenylalanine ammonia-lyases (PAL) that enhance phenylpropanoid production in planta
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