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1. Investigating the reaction and substrate preference of indole-3-acetaldehyde dehydrogenase from the plant pathogen Pseudomonas syringae Pto DC3000

   https://doi.org/10.1042/BSR20202959  

   Zhang, Kaleena; Lee, Josephine S.; Liu, Regina; Chan, Zita T.; Dawson, Trenton J.; De Togni, Elisa S.; Edwards, Chris T.; Eng, Isabel K.; Gao, Ashley R.; Goicouria, Luis A.; et al (December 2020, Bioscience Reports)

   null (Ed.)

   Abstract Aldehyde dehydrogenases (ALDHs) catalyze the conversion of various aliphatic and aromatic aldehydes into corresponding carboxylic acids. Traditionally considered as housekeeping enzymes, new biochemical roles are being identified for members of ALDH family. Recent work showed that AldA from the plant pathogen Pseudomonas syringae strain PtoDC3000 (PtoDC3000) functions as an indole-3-acetaldehyde dehydrogenase for the synthesis of indole-3-acetic acid (IAA). IAA produced by AldA allows the pathogen to suppress salicylic acid-mediated defenses in the model plant Arabidopsis thaliana. Here we present a biochemical and structural analysis of the AldA indole-3-acetaldehyde dehydrogenase from PtoDC3000. Site-directed mutants targeting the catalytic residues Cys302 and Glu267 resulted in a loss of enzymatic activity. The X-ray crystal structure of the catalytically inactive AldA C302A mutant in complex with IAA and NAD+ showed the cofactor adopting a conformation that differs from the previously reported structure of AldA. These structures suggest that NAD+ undergoes a conformational change during the AldA reaction mechanism similar to that reported for human ALDH. Site-directed mutagenesis of the IAA binding site indicates that changes in the active site surface reduces AldA activity; however, substitution of Phe169 with a tryptophan altered the substrate selectivity of the mutant to prefer octanal. The present study highlights the inherent biochemical versatility of members of the ALDH enzyme superfamily in P. syringae. 

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2. Copper binding by a unique family of metalloproteins is dependent on kynurenine formation

   https://doi.org/10.1073/pnas.2100680118  

   Manesis, Anastasia C.; Jodts, Richard J.; Hoffman, Brian M.; Rosenzweig, Amy C. (June 2021, Proceedings of the National Academy of Sciences)

   null (Ed.)

   Some methane-oxidizing bacteria use the ribosomally synthesized, posttranslationally modified natural product methanobactin (Mbn) to acquire copper for their primary metabolic enzyme, particulate methane monooxygenase. The operons encoding the machinery to biosynthesize and transport Mbns typically include genes for two proteins, MbnH and MbnP, which are also found as a pair in other genomic contexts related to copper homeostasis. While the MbnH protein, a member of the bacterial diheme cytochrome c peroxidase (bC c P)/MauG superfamily, has been characterized, the structure and function of MbnP, the relationship between the two proteins, and their role in copper homeostasis remain unclear. Biochemical characterization of MbnP from the methanotroph Methylosinus trichosporium OB3b now reveals that MbnP binds a single copper ion, present in the +1 oxidation state, with high affinity. Copper binding to MbnP in vivo is dependent on oxidation of the first tryptophan in a conserved WxW motif to a kynurenine, a transformation that occurs through an interaction of MbnH with MbnP. The 2.04-Å-resolution crystal structure of MbnP reveals a unique fold and an unusual copper-binding site involving a histidine, a methionine, a solvent ligand, and the kynurenine. Although the kynurenine residue may not serve as a Cu I primary-sphere ligand, being positioned ∼2.9 Å away from the Cu I ion, its presence is required for copper binding. Genomic neighborhood analysis indicates that MbnP proteins, and by extension kynurenine-containing copper sites, are widespread and may play diverse roles in microbial copper homeostasis. 

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3. Biochemical investigation of the tryptophan biosynthetic enzyme anthranilate phosphoribosyltransferase in plants

   https://doi.org/10.1016/j.jbc.2023.105197  

   Li, Miriam; Tadfie, Hisham; Darnell, Cameron G.; Holland, Cynthia K. (October 2023, Journal of Biological Chemistry)

   While mammals require the essential amino acid tryptophan (Trp) in their diet, plants and microorganisms synthesize Trp de novo. The five-step Trp pathway starts with the shikimate pathway product, chorismate. Chorismate is converted to the aromatic compound anthranilate, which is then conjugated to a phosphoribosyl sugar in the second step by anthranilate phosphoribosyltransferase (PAT1). As a single-copy gene in plants, all fixed carbon flux to indole and Trp for protein synthesis, specialized metabolism, and auxin hormone biosynthesis proceeds through PAT1. While bacterial PAT1s have been studied extensively, plant PAT1s have escaped biochemical characterization. Using a structure model, we identified putative active site residues that were variable across plants and kinetically characterized six PAT1s (Arabidopsis thaliana (thale cress), Citrus sinensis (sweet orange), Pistacia vera (pistachio), Juglans regia (English walnut), Selaginella moellendorffii (spike moss), and Physcomitrium patens (spreading earth-moss)). We probed the catalytic efficiency, substrate promiscuity, and regulation of these six enzymes and found that the C. sinensis PAT1 is highly specific for its cognate substrate, anthranilate. Investigations of site-directed mutants of the A. thaliana PAT1 uncovered an active site residue that contributes to promiscuity. While Trp inhibits bacterial PAT1 enzymes, the six plant PAT1s that we tested were not modu- lated by Trp. Instead, the P. patens PAT1 was inhibited by tyrosine, and the S. moellendorffii PAT1 was inhibited by phenylalanine. This structure-informed biochemical examina- tion identified variations in activity, efficiency, specificity, and enzyme-level regulation across PAT1s from evolutionarily diverse plants. 

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4. Reducing brain kynurenic acid synthesis precludes kynurenine‐induced sleep disturbances

   https://doi.org/10.1111/jsr.14038  

   Rentschler, Katherine M; Milosavljevic, Snezana; Baratta, Annalisa M; Wright, Courtney J; Piroli, Maria V; Tentor, Zachary; Valafar, Homayoun; O'Reilly, Christian; Pocivavsek, Ana (September 2023, Journal of Sleep Research)

   Summary Patients with neurocognitive disorders often battle sleep disturbances. Kynurenic acid is a tryptophan metabolite of the kynurenine pathway implicated in the pathology of these illnesses. Modest increases in kynurenic acid, an antagonist at glutamatergic and cholinergic receptors, result in cognitive impairments and sleep dysfunction. We explored the hypothesis that inhibition of the kynurenic acid synthesising enzyme, kynurenine aminotransferase II, may alleviate sleep disturbances. At the start of the light phase, adult male and female Wistar rats received systemic injections of either: (i) vehicle; (ii) kynurenine (100 mg kg⁻¹; i.p.); (iii) the kynurenine aminotransferase II inhibitor, PF‐04859989 (30 mg kg⁻¹; s.c.); or (iv) PF‐04859989 and kynurenine in combination. Kynurenine and kynurenic acid levels were evaluated in the plasma and brain. Separate animals were implanted with electroencephalogram and electromyogram telemetry devices to record polysomnography, and evaluate the vigilance states wake, rapid eye movement sleep and non‐rapid eye movement sleep following each treatment. Kynurenine challenge increased brain kynurenic acid and resulted in reduced rapid eye movement sleep duration, non‐rapid eye movement sleep delta power and sleep spindles. PF‐04859989 reduced brain kynurenic acid formation when given prior to kynurenine, prevented disturbances in rapid eye movement sleep and sleep spindles, and enhanced non‐rapid eye movement sleep. Our findings suggest that reducing kynurenic acid in conditions where the kynurenine pathway is activated may serve as a potential strategy for improving sleep dynamics. 

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5. Enzymes in a human cytoplasm model organize into submetabolon complexes

   https://doi.org/10.1073/pnas.2414206122  

   Samuel_Russell, Premila P; Rickard, Meredith M; Pogorelov, Taras V; Gruebele, Martin (February 2025, Proceedings of the National Academy of Sciences)

   Enzyme–enzyme interactions are fundamental to the function of cells. Their atomistic mechanisms remain elusive mainly due to limitations of in-cell measurements. We address this challenge by atomistically modeling, for a total of ≈80 μs, a slice of the human cell cytoplasm that includes three successive enzymes along the glycolytic pathway: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), and phosphoglycerate mutase (PGM). We tested the model for nonspecific protein stickiness, an artifact of current atomistic force fields in crowded environments. The simulations reveal that the human enzymes co-organize in-cell into transient submetabolon complexes, consistent with previous experimental results. Our data both reiterate known specificity between GAPDH and PGK and reveal extensive direct interactions between GAPDH and PGM. Our simulations further reveal, through force field benchmarking, the critical role of protein solvation in facilitating these enzyme–enzyme interactions. Transient interenzyme interactions with μs lifetime occur repeatedly in our simulations via specific sticky protein surface patches, with interactions often mediated by charged patch residues. Some of the residues that interact frequently with one another lie in or near the active site of the enzymes. We show that some of these patches correspond to a general mode to interact with several partners for promiscuous enzymes like GAPDH. We further show that the non-native yeast PGK is stickier than human PGK in our human cytoplasm model, supporting the idea of evolutionary pressure to reduce sticking. Our cytoplasm modeling paves the way toward capturing the atomistic dynamics of an entire enzymatic pathway in-cell. 

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