Designing a Novel Temperature- and Noncanonical Amino Acid-Controlled Biological Logic Gate in Escherichia coli.

Genetic code expansion (GCE) is a powerful strategy that expands the genetic code of an organism for incorporating noncanonical amino acids into proteins using engineered tRNAs and aminoacyl-tRNA synthetases (aaRSs). While GCE has opened up new possibilities for synthetic biology, little is known about the potential side effects of exogenous aaRS/tRNA pairs. In this study, we investigated the impact of exogenous aaRS and amber suppressor tRNA on gene expression in Escherichia coli. We discovered that in DH10ß ΔcyaA, transformed with the F1RP/F2P two-hybrid system, the high consumption rate of cellular adenosine triphosphate by exogenous aaRS/tRNA at elevated temperatures induces temperature sensitivity in the expression of genes regulated by the cyclic AMP receptor protein (CRP). We harnessed this temperature sensitivity to create a novel biological AND gate in E. coli, responsive to both p-benzoylphenylalanine (BzF) and low temperature, using a BzF-dependent variant of E. coli chorismate mutase and split subunits of Bordetella pertussis adenylate cyclase. Our study provides new insights into the unexpected effects of exogenous aaRS/tRNA pairs and offers a new approach for constructing a biological logic gate.

Properties and Functional Analysis of Two Chorismate Mutases from Maritime Pine.

Through the shikimate pathway, a massive metabolic flux connects the central carbon metabolism with the synthesis of chorismate, the common precursor of the aromatic amino acids phenylalanine, tyrosine, and tryptophan, as well as other compounds, including salicylate or folate. The alternative metabolic channeling of chorismate involves a key branch-point, finely regulated by aromatic amino acid levels. Chorismate mutase catalyzes the conversion of chorismate to prephenate, a precursor of phenylalanine and tyrosine and thus a vast repertoire of fundamental derived compounds, such as flavonoids or lignin. The regulation of this enzyme has been addressed in several plant species, but no study has included conifers or other gymnosperms, despite the importance of the phenolic metabolism for these plants in processes such as lignification and wood formation. Here, we show that maritime pine (Pinus pinaster Aiton) has two genes that encode for chorismate mutase, PpCM1 and PpCM2. Our investigations reveal that these genes encode plastidial isoenzymes displaying activities enhanced by tryptophan and repressed by phenylalanine and tyrosine. Using phylogenetic studies, we have provided new insights into the possible evolutionary origin of the cytosolic chorismate mutases in angiosperms involved in the synthesis of phenylalanine outside the plastid. Studies based on different platforms of gene expression and co-expression analysis have allowed us to propose that PpCM2 plays a central role in the phenylalanine synthesis pathway associated with lignification.

The influence of model building schemes and molecular dynamics sampling on QM-cluster models: the chorismate mutase case study.

Most QM-cluster models of enzymes are constructed based on X-ray crystal structures, which limits comparison to in vivo structure and mechanism. The active site of chorismate mutase from Bacillus subtilis and the enzymatic transformation of chorismate to prephenate is used as a case study to guide construction of QM-cluster models built first from the X-ray crystal structure, then from molecular dynamics (MD) simulation snapshots. The Residue Interaction Network ResidUe Selector (RINRUS) software toolkit, developed by our group to simplify and automate the construction of QM-cluster models, is expanded to handle MD to QM-cluster model workflows. Several options, some employing novel topological clustering from residue interaction network (RIN) information, are evaluated for generating conformational clustering from MD simulation. RINRUS then generates a statistical thermodynamic framework for QM-cluster modeling of the chorismate mutase mechanism via refining 250 MD frames with density functional theory (DFT). The 250 QM-cluster models sampled provide a mean ΔG‡ of 10.3 ± 2.6 kcal mol-1 compared to the experimental value of 15.4 kcal mol-1 at 25 °C. While the difference between theory and experiment is consequential, the level of theory used is modest and therefore "chemical" accuracy is unexpected. More important are the comparisons made between QM-cluster models designed from the X-ray crystal structure versus those from MD frames. The large variations in kinetic and thermodynamic properties arise from geometric changes in the ensemble of QM-cluster models, rather from the composition of the QM-cluster models or from the active site-solvent interface. The findings open the way for further quantitative and reproducible calibration in the field of computational enzymology using the model construction framework afforded with the RINRUS software toolkit.

Metabolic engineering of Synechocystis sp. PCC 6803 for the improved production of phenylpropanoids.

BACKGROUND: Phenylpropanoids are a large group of plant secondary metabolites with various biological functions, derived from aromatic amino acids. Cyanobacteria are promising host organisms for sustainable production of plant phenylpropanoids. We have previously engineered Synechocystis sp. PCC 6803 to produce trans-cinnamic acid (tCA) and p-coumaric acid (pCou), the first intermediates of phenylpropanoid pathway, by overexpression of phenylalanine- and tyrosine ammonia lyases. In this study, we aimed to enhance the production of the target compounds tCA and pCou in Synechocystis. RESULTS: We eliminated the 4-hydroxyphenylpyruvate dioxygenase (HPPD) activity, which is a competing pathway consuming tyrosine and, possibly, phenylalanine for tocopherol synthesis. Moreover, several genes of the terminal steps of the shikimate pathway were overexpressed alone or in operons, such as aromatic transaminases, feedback insensitive cyclohexadienyl dehydrogenase (TyrC) from Zymomonas mobilis and the chorismate mutase (CM) domain of the fused chorismate mutase/prephenate dehydratase enzyme from Escherichia coli. The obtained engineered strains demonstrated nearly 1.5 times enhanced tCA and pCou production when HPPD was knocked out compared to the parental production strains, accumulating 138 ± 3.5 mg L-1 of tCA and 72.3 ± 10.3 mg L-1 of pCou after seven days of photoautotrophic growth. However, there was no further improvement when any of the pathway genes were overexpressed. Finally, we used previously obtained AtPRM8 and TsPRM8 Synechocystis strains with deregulated shikimate pathway as a background for the overexpression of synthetic constructs with ppd knockout. CONCLUSIONS: HPPD elimination enhances the tCA and pCou productivity to a similar extent. The use of PRM8 based strains as a background for overexpression of synthetic constructs, however, did not promote tCA and pCou titers, which indicates a tight regulation of the terminal steps of phenylalanine and tyrosine synthesis. This work contributes to establishing cyanobacteria as hosts for phenylpropanoid production.

Accurate Computation of Thermodynamic Activation Parameters in the Chorismate Mutase Reaction from Empirical Valence Bond Simulations.

Chorismate mutase (CM) enzymes have long served as model systems for benchmarking new methods and tools in computational chemistry. Despite the enzymes' prominence in the literature, the extent of the roles that activation enthalpy and entropy play in catalyzing the conversion of chorismate to prephenate is still subject to debate. Knowledge of these parameters is a key piece in fully understanding the mechanism of chorismate mutases. Within this study, we utilize EVB/MD free energy perturbation calculations at a range of temperatures, allowing us to extract activation enthalpies and entropies from an Arrhenius plot of activation free energies of the reaction catalyzed by a monofunctional Bacillus subtilis CM and the promiscuous enzyme isochorismate pyruvate lyase of Pseudomonas aeruginosa. In comparison to the uncatalyzed reaction, our results show that both enzyme-catalyzed reactions exhibit a substantial reduction in activation enthalpy, while the effect on activation entropy is relatively minor, demonstrating that enzyme-catalyzed CM reactions are enthalpically driven. Furthermore, we observe that the monofunctional CM from B. subtilis more efficiently catalyzes this reaction than its promiscuous counterpart. This is supported by a structural analysis of the reaction pathway at the transition state, from which we identified key residues explaining the enthalpically driven nature of the reactions and also the difference in efficiencies between the two enzymes.

A secreted form of chorismate mutase (Rv1885c) in Mycobacterium bovis BCG contributes to pathogenesis by inhibiting mitochondria-mediated apoptotic cell death of macrophages.

BACKGROUND: Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), and its pathogenicity is associated with its ability to evade the host defense system. The secretory form of the chorismate mutase of M. tuberculosis (TBCM, encoded by Rv1885c) is assumed to play a key role in the pathogenesis of TB; however, the mechanism remains unknown. METHODS: A tbcm deletion mutant (B∆tbcm) was generated by targeted gene knockout in BCG to investigate the pathogenic role of TBCM in mice or macrophages. We compared the pathogenesis of B∆tbcm and wild-type BCG in vivo by measuring the bacterial clearance rate and the degree of apoptosis. Promotion of the intrinsic apoptotic pathway was evaluated in infected bone marrow-derived macrophages (BMDMs) by measuring apoptotic cell death, loss of mitochondrial membrane potential and translocation of pore-forming proteins. Immunocytochemistry, western blotting and real-time PCR were also performed to assess the related protein expression levels after infection. Furthermore, these findings were validated by complementation of tbcm in BCG. RESULTS: Deletion of the tbcm gene in BCG leads to reduced pathogenesis in a mouse model, compared to wild type BCG, by promoting apoptotic cell death and bacterial clearance. Based on these findings, we found that intrinsic apoptosis and mitochondrial impairment were promoted in B∆tbcm-infected BMDMs. B∆tbcm down-regulates the expression of Bcl-2, which leads to mitochondrial outer membrane permeabilization (MOMP), culminating in cytochrome c release from mitochondria. Consistent with this, transcriptome profiling also indicated that B∆tbcm infection is more closely related to altered mitochondrial-related gene expression than wild-type BCG infection, suggesting an inhibitory role of TBCM in mitochondrial dysfunction. Moreover, genetic complementation of B∆tbcm (C∆tbcm) restored its capacity to inhibit mitochondria-mediated apoptotic cell death. CONCLUSIONS: Our findings demonstrate the contribution of TBCM to bacterial survival, inhibiting intrinsic apoptotic cell death of macrophages as a virulence factor of M. tuberculosis complex (MTBC) strains, which could be a potential target for the development of TB therapy.

OsCM regulates rice defence system in response to UV light supplemented with drought stress.

Studies on plant responses to combined abiotic stresses are very limited, especially in major crop plants. The current study evaluated the response of chorismate mutase overexpressor (OxCM) rice line to combined UV light and drought stress. The experiments were conducted in pots in a growth chamber, and data were assessed for gene expression, antioxidant and hormone regulation, flavonoid accumulation, phenotypic variation, and amino acid accumulation. Wild-type (WT) rice had reduced the growth and vigour, while transgenic rice maintained growth and vigour under combined UV light and drought stress. ROS and lipid peroxidation analysis revealed that chorismate mutase (OsCM) reduced oxidative stress mediated by ROS scavenging and reduced lipid peroxidation. The combined stresses reduced biosynthesis of total flavonoids, kaempferol and quercetin in WT plants, but increased significantly in plants with OxCM. Phytohormone analysis showed that SA was reduced by 50% in WT and 73% in transgenic plants, while ABA was reduced by 22% in WT plants but increased to 129% in transgenic plants. Expression of chorismate mutase regulates phenylalanine biosynthesis, UV light and drought stress-responsive genes, e.g., phenylalanine ammonia lyase (OsPAL), dehydrin (OsDHN), dehydration-responsive element-binding (OsDREB), ras-related protein 7 (OsRab7), ultraviolet-B resistance 8 (OsUVR8), WRKY transcription factor 89 (OsWRKY89) and tryptophan synthase alpha chain (OsTSA). Moreover, OsCM also increases accumulation of free amino acids (aspartic acid, glutamic acid, leucine, tyrosine, phenylalanine and proline) and sodium (Na), potassium (K), and calcium (Ca) ions in response to the combined stresses. Together, these results suggest that chorismate mutase expression induces physiological, biochemical and molecular changes that enhance rice tolerance to combined UV light and drought stresses.

Best Practices on QM/MM Simulations of Biological Systems.

During the second half of the 20th century, following structural biology hallmark works on DNA and proteins, biochemists shifted their questions from "what does this molecule look like?" to "how does this process work?". Prompted by the theoretical and practical developments in computational chemistry, this led to the emergence of biomolecular simulations and, along with the 2013 Nobel Prize in Chemistry, to the development of hybrid QM/MM methods. QM/MM methods are necessary whenever the problem we want to address involves chemical reactivity and/or a change in the system's electronic structure, with archetypal examples being the studies of an enzyme's reaction mechanism and a metalloprotein's active site. In the last decades QM/MM methods have seen an increasing adoption driven by their incorporation in widely used biomolecular simulation software. However, properly setting up a QM/MM simulation is not an easy task, and several issues need to be properly addressed to obtain meaningful results. In the present work, we describe both the theoretical concepts and practical issues that need to be considered when performing QM/MM simulations. We start with a brief historical perspective on the development of these methods and describe when and why QM/MM methods are mandatory. Then we show how to properly select and analyze the performance of the QM level of theory, the QM system size, and the position and type of the boundaries. We show the relevance of performing prior QM model system (or QM cluster) calculations in a vacuum and how to use the corresponding results to adequately calibrate those derived from QM/MM. We also discuss how to prepare the starting structure and how to select an adequate simulation strategy, including those based on geometry optimizations as well as free energy methods. In particular, we focus on the determination of free energy profiles using multiple steered molecular dynamics (MSMD) combined with Jarzynski's equation. Finally, we describe the results for two illustrative and complementary examples: the reaction performed by chorismate mutase and the study of ligand binding to hemoglobins. Overall, we provide many practical recommendations (or shortcuts) together with important conceptualizations that we hope will encourage more and more researchers to incorporate QM/MM studies into their research projects.

Wang resin catalysed sonochemical synthesis of pyrazolo[4,3-d]pyrimidinones and 2,3-dihydroquinazolin-4(1H)-ones: Identification of chorismate mutase inhibitors having effects on Mycobacterium tuberculosis cell viability.

The enzyme chorismate mutase (or CM that is vital for the survival of bacteria) is an interesting pharmacological target for the identification of new anti-tubercular agents. The 5,5-disibstituted pyrazolo[4,3-d]pyrimidinone derivatives containing the fragment based on 4-amino-1-methyl-3-propyl-1H-pyrazole-5-carboxamide were designed and explored as the potential inhibitors of chorismate mutase. Based on encouraging docking results of two representative molecules evaluated in silico against MtbCM (PDB: 2FP2) the Wang resin catalysed sonochemical synthesis of target N-heteroarenes were undertaken. The methodology involved the reaction of 4-amino-1-methyl-3-propyl-1H-pyrazole-5-carboxamide with the appropriate cyclic/acyclic ketones to afford the desired products in acceptable (51-94%) yields. The methodology was also extended successfully towards the synthesis of 2,2-disubstituted 2,3-dihydroquinazolin-4(1H)-ones in excellent (85-90%) yields. In vitro MTT assay against the RAW 264.7 cell line followed by enzymatic assay against MtbCM identified 3b and 3c as active compounds that showed two H-bonding via their NH (at position 6) and CO group with MtbCM in silico and encouraging (54-57%) inhibition at 30 µM in vitro. Notably, none of the 2,2-disubstituted 2,3-dihydroquinazolin-4(1H)-ones showed any significant inhibition of MtbCM suggesting the favourable role of the pyrazole moiety in case of pyrazolo[4,3-d]pyrimidinones. The favourable role of cyclopentyl ring attached to the pyrazolo[4,3-d]pyrimidinone moiety and that of two methyl groups in place of cyclopentyl ring was also indicated by the SAR study. Besides showing effects against MtbCM in the concentration response study, 3b and 3c showed little or no effects on mammalian cell viability up to 100 µM in an MTT assay but decreased the % Mtb cell viability at 10-30 µM with > 20% decrease at 30 µM in an Alamar Blue Assay. Moreover, no adverse effects were noted for these compounds when tested for teratogenicity and hepatotoxicity in zebrafish at various concentrations. Overall, being the only example of MtbCM inhibitors that showed effects on Mtb cell viability the compound 3b and 3c are of further interest form the view point of discovery and development of new anti-tubercular agents.

Large language models generate functional protein sequences across diverse families.

Deep-learning language models have shown promise in various biotechnological applications, including protein design and engineering. Here we describe ProGen, a language model that can generate protein sequences with a predictable function across large protein families, akin to generating grammatically and semantically correct natural language sentences on diverse topics. The model was trained on 280 million protein sequences from >19,000 families and is augmented with control tags specifying protein properties. ProGen can be further fine-tuned to curated sequences and tags to improve controllable generation performance of proteins from families with sufficient homologous samples. Artificial proteins fine-tuned to five distinct lysozyme families showed similar catalytic efficiencies as natural lysozymes, with sequence identity to natural proteins as low as 31.4%. ProGen is readily adapted to diverse protein families, as we demonstrate with chorismate mutase and malate dehydrogenase.

What Drives Chorismate Mutase to Top Performance? Insights from a Combined In Silico and In Vitro Study.

Unlike typical chorismate mutases, the enzyme from Mycobacterium tuberculosis (MtCM) has only low activity on its own. Remarkably, its catalytic efficiency kcat/Km can be boosted more than 100-fold by complex formation with a partner enzyme. Recently, an autonomously fully active MtCM variant was generated using directed evolution, and its structure was solved by X-ray crystallography. However, key residues were involved in crystal contacts, challenging the functional interpretation of the structural changes. Here, we address these challenges by microsecond molecular dynamics simulations, followed up by additional kinetic and structural analyses of selected sets of specifically engineered enzyme variants. A comparison of wild-type MtCM with naturally and artificially activated MtCMs revealed the overall dynamic profiles of these enzymes as well as key interactions between the C-terminus and the active site loop. In the artificially evolved variant of this model enzyme, this loop is preorganized and stabilized by Pro52 and Asp55, two highly conserved residues in typical, highly active chorismate mutases. Asp55 stretches across the active site and helps to appropriately position active site residues Arg18 and Arg46 for catalysis. The role of Asp55 can be taken over by another acidic residue, if introduced at position 88 close to the C-terminus of MtCM, as suggested by molecular dynamics simulations and confirmed by kinetic investigations of engineered variants.

De novo design of anti-tuberculosis agents using a structure-based deep learning method.

Mycobacterium tuberculosis (Mtb) is a pathogen of major concern due to its ability to withstand both first- and second-line antibiotics, leading to drug resistance. Thus, there is a critical need for identification of novel anti-tuberculosis agents targeting Mtb-specific proteins. The ceaseless search for novel antimicrobial agents to combat drug-resistant bacteria can be accelerated by the development of advanced deep learning methods, to explore both existing and uncharted regions of the chemical space. The adaptation of deep learning methods to under-explored pathogens such as Mtb is a challenging aspect, as most of the existing methods rely on the availability of sufficient target-specific ligand data to design novel small molecules with optimized bioactivity. In this work, we report the design of novel anti-tuberculosis agents targeting the Mtb chorismate mutase protein using a structure-based drug design algorithm. The structure-based deep learning method relies on the knowledge of the target protein's binding site structure alone for conditional generation of novel small molecules. The method eliminates the need for curation of a high-quality target-specific small molecule dataset, which remains a challenge even for many druggable targets, including Mtb chorismate mutase. Novel molecules are proposed, that show high complementarity to the target binding site. The graph attention model could identify the probable key binding site residues, which influenced the conditional molecule generator to design new molecules with pharmacophoric features similar to the known inhibitors.

Mechanism of chorismate dehydratase MqnA, the first enzyme of the futalosine pathway, proceeds via substrate-assisted catalysis.

MqnA, the only chorismate dehydratase known so far, catalyzes the initial step in the biosynthesis of menaquinone via the futalosine pathway. Details of the MqnA reaction mechanism remain unclear. Here, we present crystal structures of Streptomyces coelicolor MqnA and its active site mutants in complex with chorismate and the product 3-enolpyruvyl-benzoate, produced during heterologous expression in Escherichia coli. Together with activity studies, our data are in line with dehydration proceeding via substrate assisted catalysis, with the enol pyruvyl group of chorismate acting as catalytic base. Surprisingly, structures of the mutant Asn17Asp with copurified ligand suggest that the enzyme converts to a hydrolase by serendipitous positioning of the carboxyl group. All complex structures presented here exhibit a closed Venus flytrap fold, with the enzyme exploiting the characteristic ligand binding properties of the fold for specific substrate binding and catalysis. The conformational rearrangements that facilitate complete burial of substrate/product, with accompanying topological changes to the enzyme surface, could foster substrate channeling within the biosynthetic pathway.

Conformational Transitions in Yeast Chorismate Mutase Important for Allosteric Regulation as Identified by Nuclear Magnetic Resonance Spectroscopy.

Proteins fluctuate between different conformations in solution, and these conformational fluctuations can be important for protein function and allosteric regulation. The chorismate mutase from Saccharomyces cerevisiae (ScCM), a key enzyme in the biosynthesis of aromatic amino acids, is allosterically activated and inhibited by tryptophan and tyrosine, respectively. It was initially proposed that in the absence of effector, ScCM fluctuates between activated R and inhibited T conformations according to the Monod-Wyman-Changeux (MWC) model, although a more complex regulation pattern was later suggested by mutagenesis and kinetic data. Here we used NMR relaxation dispersion experiments to understand the conformational fluctuations on the microsecond-to-millisecond timescale that occur in ScCM. In the absence of allosteric effectors, ScCM did not exclusively exchange between T and R conformations, suggesting that the two-state MWC model is insufficient to explain conformational dynamics. Addition of tyrosine led to the quenching of much of the motion on this timescale, while new motions were identified in the presence of tryptophan. These new motions are consistent with conformational fluctuations into an alternative conformation that may be important for enzyme activity.

Potential of Mycobacterium tuberculosis chorismate mutase (Rv1885c) as a novel TLR4-mediated adjuvant for dendritic cell-based cancer immunotherapy.

For clinical application by dendritic cell (DC)-based cancer immunotherapy, a proper adjuvant system to elicit a strong anticancer immune response is needed. Here, we investigated the potential of chorismate mutase (TBCM, Rv1885c), a putative Mycobacterium tuberculosis (TB) virulence factor, as an immunoadjuvant in DC-based tumor immunotherapy. First, we found that TBCM functionally activated DCs by upregulating costimulatory molecules, increasing the secretion of proinflammatory cytokines, enhancing migration and inducing the Th1-type immune response in a dose-dependent manner via TLR4-mediated signaling. In addition, subcutaneous injection of TBCM-activated DCs loaded with cell lysates led to reduced tumor mass, enhanced mouse survival and lowered tumor incidence in lung carcinoma (LLC) cell-bearing mice. This is mainly mediated by functional cytotoxic T lymphocyte-mediated oncolytic activity and inhibition of cancer proliferation- and metastasis-related genes. Moreover, TBCM-induced DCs can also generate memory CD4 T cells and exert long-term tumor prevention effects. In conclusion, our findings suggest that TBCM (Rv1885c), a novel TLR4 agonist, could be used as an immunoadjuvant for DC-based cancer immunotherapy.

Can the local electric field be a descriptor of catalytic activity? A case study on chorismate mutase.

The current theoretical perception of enzymatic activity is highly reliant on the determination of the activation energy of the reactions, which is often calculated using computationally demanding quantum mechanical calculations. With the ever-increasing use of bioengineering techniques that produce too many variants of the same enzyme, a fast and accurate way to study the relative efficiency of enzymes is currently in high demand. Here, we propose the local electric field (LEF) of the enzyme along the reaction axis as a descriptor for the enzymatic activity using the example of chorismate mutase in its native form and several variants (R90A, R90G, and R90K/C88S). The study shows a direct correlation between the calculated enzymatic EF and the enzymatic activity for all the complexes. MD simulations of the Michaelis complex and the transition state analog (TSA) show a stabilizing force on the TSA due to the enzymatic EF. QM/MM and QM-only DFT calculations in the presence of an external electric field (EEF) oriented along the reaction axis show that the electric field can interact with the dipole moment of the TS, thereby stabilizing it and thus lowering the activation energy.

A Secreted Chorismate Mutase from Xanthomonas arboricola pv. juglandis Attenuates Virulence and Walnut Blight Symptoms.

Walnut blight is a significant above-ground disease of walnuts caused by Xanthomonas arboricola pv. juglandis (Xaj). The secreted form of chorismate mutase (CM), a key enzyme of the shikimate pathway regulating plant immunity, is highly conserved between plant-associated beta and gamma proteobacteria including phytopathogens belonging to the Xanthomonadaceae family. To define its role in walnut blight disease, a dysfunctional mutant of chorismate mutase was created in a copper resistant strain Xaj417 (XajCM). Infections of immature walnut Juglans regia (Jr) fruit with XajCM were hypervirulent compared with infections with the wildtype Xaj417 strain. The in vitro growth rate, size and cellular morphology were similar between the wild-type and XajCM mutant strains, however the quantification of bacterial cells by dPCR within walnut hull tissues showed a 27% increase in XajCM seven days post-infection. To define the mechanism of hypervirulence, proteome analysis was conducted to compare walnut hull tissues inoculated with the wild type to those inoculated with the XajCM mutant strain. Proteome analysis revealed 3296 Jr proteins (five decreased and ten increased with FDR ≤ 0.05) and 676 Xaj417 proteins (235 increased in XajCM with FDR ≤ 0.05). Interestingly, the most abundant protein in Xaj was a polygalacturonase, while in Jr it was a polygalacturonase inhibitor. These results suggest that this secreted chorismate mutase may be an important virulence suppressor gene that regulates Xaj417 virulence response, allowing for improved bacterial survival in the plant tissues.

Chorismate mutase peptide antibody enables specific detection of Acanthamoeba.

Accurate and rapid diagnosis of Acanthamoeba keratitis (AK) is difficult. Although the diagnostic procedure for AK has improved, further development and effective diagnostic tool utilization for AK need to continue. Chorismate mutase is a key regulatory enzyme involved in the shikimate pathway, a metabolic pathway absent in mammals but central for amino acid biosynthesis in bacteria, fungi, algae, and plants. In this study, we describe the identification and production of a polyclonal peptide antibody targeting chorismate mutase secreted by A. castellanii, which could be used for AK diagnosis. Western blot was performed using the protein lysates and conditioned media of the human corneal epithelial (HCE) cells, non-pathogenic Acanthamoeba, pathogenic Acanthamoeba, clinical isolate of Acanthamoeba spp., and other causes of keratitis such as Fusarium solani, Pseudomonas aeruginosa, and Staphylococcus aureus. Polyclonal antibodies raised against A. castellanii chorismate mutase specifically interacted with lysates of Acanthamoeba origin and their culture media, while such interactions were not observed from other samples. Acanthamoeba-specificity of chorismate mutase was also confirmed using immunocytochemistry after co-culturing Acanthamoeba with HCE cells. Specific binding of the chorismate mutase antibody to Acanthamoeba was observed, which were absent in the case of HCE cells. These results indicate that the chorismate mutase antibody of Acanthamoeba may serve as a method for rapid and differential Acanthamoeba identification.

Unravelling the Allosteric Targeting of PHGDH at the ACT-Binding Domain with a Photoactivatable Diazirine Probe and Mass Spectrometry Experiments.

The serine biosynthetic pathway is a key element contributing to tumor proliferation. In recent years, targeting of phosphoglycerate dehydrogenase (PHGDH), the first enzyme of this pathway, intensified and revealed to be a promising strategy to develop new anticancer drugs. Among attractive PHGDH inhibitors are the α-ketothioamides. In previous work, we have demonstrated their efficacy in the inhibition of PHGDH in vitro and in cellulo. However, the precise site of action of this series, which would help the rational design of new inhibitors, remained undefined. In the present study, the detailed mechanism-of-action of a representative α-ketothioamide inhibitor is reported using several complementary experimental techniques. Strikingly, our work led to the identification of an allosteric site on PHGDH that can be targeted for drug development. Using mass spectrometry experiments and an original α-ketothioamide diazirine-based photoaffinity probe, we identified the 523Q-533F sequence on the ACT regulatory domain of PHGDH as the binding site of α-ketothioamides. Mutagenesis experiments further documented the specificity of our compound at this allosteric site. Our results thus pave the way for the development of new anticancer drugs using a completely novel mechanism-of-action.

A novel chorismate mutase from Erysiphe quercicola performs dual functions of synthesizing amino acids and inhibiting plant salicylic acid synthesis.

Pathogens secrete effectors to establish a successful interaction with their host. It is well understood that plant pathogens recruit classically secreted chorismate mutase (Cmu) as an effector to disrupt plant salicylic acid (SA) synthesis. However, the identity and function of the Cmu effector from powdery mildew fungi remain unknown. Here, we identified a novel secreted Cmu effector, EqCmu, from rubber (Hevea brasiliensis Muell) powdery mildew fungus (Erysiphe quercicola). Unlike the classically secreted Cmu, EqCmu lack signal peptide, and exhibited characteristics of non-classically secreted proteins. EqCmu could fully complement a Saccharomyces cerevisiae ScAro7 mutant that was deficient in the synthesis of phenylalanine and tyrosine. In addition, transient expression of EqCmu could promote infection by Phytophthora capsici and reduce the levels of SA and the mRNA of PR1 gene in Nicotiana benthamiana in response to P. capsici infection, while confocal observations showed that EqCmu was localized within the cytoplasm and nucleus of transfected N. benthamiana leaf cells. These non-homologous systems assays provide evidences that EqCmu may serve as a "moonlighting" protein, which is not only a key enzyme in the synthesis of phenylalanine and tyrosine within fungal cells, but also has the function of regulating plant SA synthesis within plant cells. This is the first study to identify and functionally validate a candidate effector from E. quercicola. Overall, the non-classical secretion pathway is a novel mechanism for powdery mildew fungal effectors secretion and might play an important role in host-pathogen interactions.