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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.

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. [ABSTRACT FROM AUTHOR]

In the process of infecting plants, plant parasitic nematodes release a series of proteins that play an essential role in the successful infection and pathogenesis of plant cells and tissues through stylets or body walls. In this study, based on transcriptome data, a chorismate mutase gene of Radopholus similis (RsCM) was identified and cloned, which is a single copy gene specifically expressed in the oesophageal gland and highly expressed in juveniles and females. Transient expression of RsCM in tobacco leaves showed that it was localised in the cytoplasm and nucleus of tobacco leaf cells, which inhibited the pattern-triggered immunity (PTI) induced by flg22, including callose deposition and defence gene expression, and cell death induced by immune elicitors BAX, but could not inhibit cell death induced by immune elicitors Gpa2/RBP-1. The RNA interference (RNAi) transgenic tomato of RsCM obviously inhibited the infection, pathogenicity, and reproduction of R. similis. However, the resistance of the overexpression transgenic tomato of RsCM to R. similis infection was significantly reduced, and the expression levels of two salicylic acid (SA) pathway genes (PR1 and PR5) in roots infected by the nematode were significantly down-regulated, which indicated that RsCM might be involved in the inhibition of SA pathway. The results of this study demonstrate that RsCM suppresses the host immune system and might be a new target for the control of R. similis, which also provides new data for the function and mechanism of CM genes of migratory parasitic plant nematodes.

The Wang‐OSO3H catalyzed synthesis of a series of spiroindoline‐3,2'‐quinazoline derivatives and their in silico and in vitro evaluation against MtbCM (Mycobacterium tuberculosis Chorismate Mutase) are disclosed. Initially, a library of small molecules based on spiroindoline‐3,2'‐quinazoline framework was constructed. The reaction of 2‐aminobenzamide with appropriate isatin derivatives in the presence of Wang‐OSO3H under ultrasound irradiation was used for this purpose. The reaction proceeded under mild conditions to afford the desired products in good yields within a short duration. With the use of heterogeneous catalyst, inexpensive solvent as reaction media, and ultrasound as the source of green energy the methodology appeared to be a useful alternative to the previously reported methods. The in silico docking of all the synthesized compounds into the MtbCM (PDB: 2FP2) revealed that the binding of these compounds took place on the external surface pockets of the MtbCM active site cavity mainly in two different clusters. Three compounds that interacted well with MtbCM also showed good (57–64 %) inhibition of MtbCM in vitro when tested at 10 μM. Based on in silico and in vitro studies along with the ADME predictions these three spiroindoline‐3,2'‐quinazoline derivatives were identified as pre‐hits. [ABSTRACT FROM AUTHOR]

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.

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.

The soybean cyst nematode, Heterodeara glycines, is a serious pathogen of soybean, and reported to be the host of a wide range of Fabaceae. In the present study, the host specificity and reproductivity of two populations of H. glycines collected from soybean and tobacco were identified and characterized. The comparative identity between β-1,4-endoglucanase, pectate lyase and chorismate mutase of H. glycines parasitizing on soybean and tobacco were 99, 97 and 98%, respectively. The qRT-PCR analysis indicated that the expression of pectate lyase 2 gene was significantly higher in second-stage juveniles of H. glycines Henan population parasitizing on tobacco than that of H. glycines Shanxi population parasitizing on soybean. In addition, the pectic acid content of cell wall was significantly higher (45%) in the roots of tobacco than the roots of soybean. Our results indicate that the changes in transcript parasitism genes may be a result of long-term evolution illustrating how a plant-parasitic nematode adapts to the host environment for optimal infestation and survival.

The dual Hamiltonian free energy perturbation (DH-FEP) method is designed for accurate and efficient evaluation of the free energy profile of chemical reactions in quantum mechanical/molecular mechanical (QM/MM) calculations. In contrast to existing QM/MM FEP variants, the QM region is not kept frozen during sampling, but all degrees of freedom except for the reaction coordinate are sampled. In the DH-FEP scheme, the sampling is done by semiempirical QM/MM molecular dynamics (MD), while the perturbation energy differences are evaluated from high-level QM/MM single-point calculations at regular intervals, skipping a pre-defined number of MD sampling steps. After validating our method using an analytic model potential with an exactly known solution, we report a QM/MM DH-FEP study of the enzymatic reaction catalyzed by chorismate mutase. We suggest guidelines for QM/MM DH-FEP calculations and default values for the required computational parameters. In the case of chorismate mutase, we apply the DH-FEP approach in combination with a single one-dimensional reaction coordinate and with a two-dimensional collective coordinate (two individual distances), with superior results for the latter choice. [ABSTRACT FROM AUTHOR]

The crystal structures of BB2672 and SPO0826 were determined to resolutions of 1.7 and 2.1 Å by single-wavelength anomalous dispersion and multiple-wavelength anomalous dispersion, respectively, using the semi-automated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). These proteins are the first structural representatives of the PF06684 (DUF1185) Pfam family. Structural analysis revealed that both structures adopt a variant of the Bacillus chorismate mutase fold (BCM). The biological unit of both proteins is a hexamer and analysis of homologs indicates that the oligomer interface residues are highly conserved. The conformation of the critical regions for oligomerization appears to be dependent on pH or salt concentration, suggesting that this protein might be subject to environmental regulation. Structural similarities to BCM and genome-context analysis suggest a function in amino-acid synthesis.

Hirschmanniella oryzae is one of the most devastating nematodes on rice, leading to substantial yield losses. Effector proteins aid the nematode during the infection process by subduing plant defence responses. In this research we characterized two potential H. oryzae effector proteins, chorismate mutase (HoCM) and isochorismatase (HoICM), and investigated their enzymatic activity and their role in plant immunity. Both HoCM and HoICM proved to be enzymatically active in complementation tests in mutant Escherichia coli strains. Infection success by the migratory nematode H. oryzae was significantly higher in transgenic rice lines constitutively expressing HoCM or HoICM. Expression of HoCM, but not HoICM, increased rice susceptibility against the sedentary nematode Meloidogyne graminicola also. Transcriptome and metabolome analyses indicated reductions in secondary metabolites in the transgenic rice plants expressing the potential nematode effectors. The results presented here demonstrate that both HoCM and HoICM suppress the host immune system and that this may be accomplished by lowering secondary metabolite levels in the plant. [ABSTRACT FROM AUTHOR]

The rational design of enzymes is an important goal for both fundamental and practical reasons. Here, we describe a process to learn the constraints for specifying proteins purely from evolutionary sequence data, design and build libraries of synthetic genes, and test them for activity in vivo using a quantitative complementation assay. For chorismate mutase, a key enzyme in the biosynthesis of aromatic amino acids, we demonstrate the design of natural-like catalytic function with substantial sequence diversity. Further optimization focuses the generative model toward function in a specific genomic context. The data show that sequence-based statistical models suffice to specify proteins and provide access to an enormous space of functional sequences. This result provides a foundation for a general process for evolution-based design of artificial proteins. [ABSTRACT FROM AUTHOR]

• N -unsubstituted 1,2-benzothiazine 1,1-dioxides were explored against Mtb CM. • Their sonochemical synthesis involved Pd–catalyzed one-pot coupling-cyclization. • In silico studies indicated 3d, 3e and 3f as molecules of potential interest. • They formed H-bonds with GLU68, SER70 and GLY71 residues of Mtb CM. • In silico prediction indicated a good overall ADME for 3a, 3b and 3n. The Pd-catalyzed one-pot sonochemical synthesis followed by in silico and in vitro evaluation of a range of N -unsubstituted 1,2-benzothiazine 1,1-dioxide derivatives is reported. The synthesis involved ultrasound assisted coupling-cyclization of 2-iodobenzenesulfonamide with terminal alkynes in the presence of (PPh 3) 4 Pd, CuI, ZnCl 2 and Et 3 N to afford the expected products in 73-80 % yield. This is the first example of accessing N -unsubstituted 1,2-benzothiazine 1,1-dioxides via Pd-catalyzed coupling-cyclization strategy in a single pot. Moreover, the use of mild conditions and ultrasound as the source of green energy are the main features of this approach. In silico studies suggested that all the synthesized compounds interacted with the loop near the active site of Mtb chorismate mutase or Mtb CM. Indeed, these compounds showed H-bonding with residues in the hinge region of the active site loop and the benzothiazine 1,1-dioxide moiety was responsible for H-bonding with GLU68, SER70 and GLY71 residues. Three compounds e.g. 3d, 3e and 3f interacted well with Mtb CM via the aforementioned H-bonds and their orientations seemed to be influenced considerably by the effective non-H-bond interactions with LEU65 and PRO66. They also showed encouraging (54-62 %) inhibition of Mtb CM in vitro when tested at 10 µM. The outcome of in silico and in vitro studies along with the ADME predictions identified compound 3c, 3d and 3e as pre-hits for further pharmacological study. N -Unsubstituted 1,2-benzothiazine 1,1-dioxides accessed via the transition metal catalyzed one-pot sonochemical approach were explored against Mtb CM in silico and in vitro [Display omitted] [ABSTRACT FROM AUTHOR]

The Bacillus subtilis chorismate mutase catalysed Claisen rearrangement of chorismate to prephenate is one of the few pericyclic processes in biology, and as such provides a rare opportunity for understanding how Nature promotes such rearrangements so successfully. The major focus of this work is on (i) Exploring the hypothesis that the mechanism of the chorismate rearrangement is the same in the gas phase, in the aqueous solution and in the enzyme; (ii) Investigating current suggestions that the enzyme lowers the barrier via transition state stabilisation rather than via space confinement; and (iii) A comparison of Nature's way of catalysing the reaction with a gold(I) catalysed chorismate rearrangement. Based the Unified Reaction Valley Approach (URVA), for the first time, a detailed one-to-one comparison of the rearrangement in the gas phase, in the aqueous solution and in the enzyme is presented. URVA confirms that the actual chemical process of CO bond breaking and CC bond forming is the same for all media and unravels the unique catalytic function of the enzyme as a combination of shortening the process of positioning the enolpyruvyl side chain over the cyclohexadienyl ring by space confinement in concert with facilitating CO cleavage by enhanced charge polarisation. The transition state does not play a signifiant role for the rearrangement. In contrast, the gold catalyst changes the chemical process. The rearrangement is split into two steps by switching between Au[I]-π and Au[I]-σ complexation, thus avoiding the energy consuming CO breakage in the first step. Suggestions are made for metalloenzyme analogues combining both strategies. [ABSTRACT FROM AUTHOR]

Activities of phenylalanine (Phe) biosynthetic enzymes chorismate mutase (CM) and arogenate dehydratase (ADT) and of phenylalanine ammonia lyase [PAL, an enzyme that directs Phe towards capsaicinoid (CAP) synthesis] were analyzed during Capsicum chinense Jacq. (habanero pepper) fruit development. A maximum CM activity coincided with a maximum CAP accumulation. However, ADT exhibited two activity peaks, one during the early phase (10 - 17 days post-anthesis, DPA) and another during the late phase (35 - 37 DPA); only the latter coincided with CAP. Interestingly, PAL activity was inversely related to CAP accumulation; lower activities coincided with a maximum CAP content. These results suggest the operation of a control mechanism that coordinated Phe synthesis and its channeling towards CAP synthesis during the course of fruit development.

The tomato (Solanum lycopersicum) fruit is an excellent source of antioxidants, dietary fibers, minerals and vitamins and therefore has been referred to as a “functional food”. Ripe tomato fruits produce a large number of specialized metabolites including volatile organic compounds. These volatiles serve as key components of the tomato fruit flavor, participate in plant pathogen and herbivore defense, and are used to attract seed dispersers. A major class of specialized metabolites is derived from the shikimate pathway followed by aromatic amino acid biosynthesis of phenylalanine, tyrosine and tryptophan. We attempted to modify tomato fruit flavor by overexpressing key regulatory genes in the shikimate pathway. Bacterial genes encoding feedback-insensitive variants of 3-Deoxy-D-Arabino-Heptulosonate 7-Phosphate Synthase (DAHPS; AroG209-9) and bi-functional Chorismate Mutase/Prephenate Dehydratase (CM/PDT; PheA12) were expressed under the control of a fruit-specific promoter. We crossed these transgenes to generate tomato plants expressing both the AroG209 and PheA12 genes. Overexpression of the AroG209-9 gene had a dramatic effect on the overall metabolic profile of the fruit, including enhanced levels of multiple volatile and non-volatile metabolites. In contrast, the PheA12 overexpression line exhibited minor metabolic effects compared to the wild type fruit. Co-expression of both the AroG209-9 and PheA12 genes in tomato resulted overall in a similar metabolic effect to that of expressing only the AroG209-9 gene. However, the aroma ranking attributes of the tomato fruits from PheA12//AroG209-9 were unique and different from those of the lines expressing a single gene, suggesting a contribution of the PheA12 gene to the overall metabolic profile. We suggest that expression of bacterial genes encoding feedback-insensitive enzymes of the shikimate pathway in tomato fruits provides a useful metabolic engineering tool for the modification of fruits aroma and the generation of new combinations of tomato flavors.

How enzymes behave in cells is likely different from how they behave in the test tube. Previous in vitro studies find that osmolytes interact weakly with folate. Removal of the osmolyte from the solvation shell of folate is more difficult than removal of water, which weakens binding of folate to its enzyme partners. To examine if this phenomenon occurs in vivo, osmotic stress titrations were performed in Escherichia coli. Two strategies were employed: resistance to an antibacterial drug or complementation of a knockout strain by the appropriate gene cloned in a plasmid that allows tight control of expression levels as well as labeling by a degradation tag. The abilities of the knockout and complemented strains to grow under osmotic stress were compared. Typically, the knockout strain could grow to high osmolalities on supplemented media while the complemented strain stopped growing at lower osmolalities on minimal media. This pattern was observed for an R67 dihydrofolate reductase clone rescuing a ÄfolA strain, for a methylenetetrahydrofolate reductase clone rescuing a ÄmetF strain and for a serine hydroxymethyltransferase clone rescuing a ÄglyA strain. Additionally, an R67 dihydrofolate reductase clone allowed E. coli DH5á to grow in the presence of trimethoprim until ~0.81 Osm while cells in a control titration lacking antibiotic could grow until 1.90 Osm. [ABSTRACT FROM AUTHOR]

Meloidogyne incognita is the most economically important plant‐parasitic nematode. Meloidogyne incognita manipulates plant cell development and metabolism by injecting effectors from the oesophageal glands into the plant host. Chorismate mutase (CM) is one such effector that may contribute to successful parasitism by M. incognita. This investigation identified and functionally characterized a novel CM effector, Mi‐CM‐3, which is more similar to CMs from bacteria than from other phytoparasitic nematodes. Spatial and temporal expression assays showed Mi‐cm‐3 mRNA accumulates specifically in the subventral oesophageal glands and transcription is up‐regulated during the early parasitic stages of the nematode. In planta gene silencing of Mi‐cm‐3 attenuated nematode parasitism. In addition, Mi‐cm‐3 could fully restore the full virulence phenotype of the pathogenic bacterium Xanthomonas oryzae pv. oryzae by complementation when it was introduced into a mutant strain carrying a deletion in the CM gene. Transient expression of Mi‐CM‐3 caused a reduction in levels of salicylic acid (SA) and mRNA of gene PR1 in Nicotiana benthamiana in response to oomycete pathogen Phytophthora capsici infection, while confocal observations showed that Mi‐CM‐3 was localized within the cytoplasm and the nucleus, but not the plastids, of transfected N. benthamiana leaf cells. Constitutive expression of Mi‐CM‐3 in N. benthamiana plants inhibited root growth and increased susceptibility to M. incognita infection. These results provide direct experimental evidence to show that Mi‐CM‐3 may play an important role in suppressing plant immunity by regulating the SA pathway during the early parasitic stage of M. incognita so as to promote nematode parasitism. [ABSTRACT FROM AUTHOR]

Burkholderia thailandensis is often used as a model for more virulent members of this genus of proteobacteria that are highly antibiotic‐resistant and are potential agents of biological warfare that are infective by inhalation. As part of ongoing efforts to identify potential targets for the development of rational therapeutics, the structures of enzymes that are absent in humans, including that of chorismate mutase from B. thailandensis, have been determined by the Seattle Structural Genomics Center for Infectious Disease. The high‐resolution structure of chorismate mutase from B. thailandensis was determined in the monoclinic space group P21 with three homodimers per asymmetric unit. The overall structure of each protomer has the prototypical AroQγ topology and shares conserved binding‐cavity residues with other chorismate mutases, including those with which it has no appreciable sequence identity. [ABSTRACT FROM AUTHOR]

Abstract: l‐Phenylalanine is an important amino acid that is widely used in the production of food flavors and pharmaceuticals. Generally, l‐phenylalanine production by engineered Escherichia coli requires a high rate of oxygen supply. However, the coexpression of Vitreoscilla hemoglobin gene (vgb), driven bya tac promoter, with the genes encoding 3‐deoxy‐ d‐arabinoheptulosonate‐7‐phosphate synthetase (aroF) and feedback‐resistant chorismate mutase/prephenate dehydratase (pheAfbr), led to increased productivity and decreased demand for aeration by E. coli CICC10245. Shake‐flask studies showed that vgb‐expressing strains displayed higher rates of oxygen uptake, and l‐phenylalanine production under standard aeration conditions was increased. In the aerobic fermentation process, cell growth, l‐phenylalanine production, and glucose consumption by the recombinant E. coli strain PAPV, which harbored aroF, pheAfbr, and tac‐vgb genes, were increased compared to that in the strain harboring only aroF and pheAfbr (E. coli strain PAP), especially under oxygen‐limited conditions. The vgb‐expressing strain PAPV produced 21.9% more biomass and 16.6% more l‐phenylalanine, while consuming only approximately 5% more glucose after 48 H of fermentation. This study demonstrates a method to enhance the l‐phenylalanine production by E. coli using less intensive and thus more economical aeration conditions. [ABSTRACT FROM AUTHOR]

The bacterium Burkholderia phymatum is a promiscuous symbiotic nitrogen‐fixating bacterium that belongs to one of the largest groups of Betaproteobacteria. Other Burkholderia species are known to cause disease in plants and animals, and some are potential agents for biological warfare. Structural genomics efforts include characterizing the structures of enzymes from pathways that can be targeted for drug development. As part of these efforts, chorismate mutase from B. phymatum was produced and crystallized, and a 1.95 Å resolution structure is reported. This enzyme shares less than 33% sequence identity with other homologs of known structure. There are two classes of chorismate mutase: AroQ and AroH. The bacterial subclass AroQγ has reported roles in virulence. Chorismate mutase from B. phymatum has the prototypical AroQγ topology and retains the characteristic chorismate mutase active site. This suggests that substrate‐based chorismate mutase inhibitors will not be specific and are likely to affect beneficial bacteria such as B. phymatum. [ABSTRACT FROM AUTHOR]

[Display omitted] Promysalin is an amphipathic antibiotic isolated from Pseudomonas promysalinigenes (previously Pseudomonas putida RW10S1) which shows potent antibacterial activities against Gram-negative pathogens by inactivating succinate dehydrogenase. Based on the in-vivo studies, promysalin is hypothesized to be assembled from three building blocks: salicylic acid, proline, and myristic acid via a proposed but uncharacterized hybrid NRPS-PKS biosynthetic pathway. So far, no in-vitro biosynthetic studies have been reported for this promising antibiotic. Here, we report the first in-vitro reconstitution and biochemical characterization of two early enzymes on the pathway: PpgH, an isochorismate synthase (IS), and PpgG, an isochorismate pyruvate lyase (IPL) which are involved in the biosynthesis of salicylic acid, the polar fragment of promysalin. We also report a secondary chorismate mutase (CM) activity for PpgG. Based on our biochemical experiments, preliminary mechanistic proposals have been postulated for PpgH and PpgG. We believe this study will lay a strong foundation for elucidating the functions and mechanisms of other intriguing enzymes of the promysalin biosynthesis pathway, which may potentially unravel interesting enzyme chemistries and promote pathway engineering in the future. [ABSTRACT FROM AUTHOR]

Both drought and salinity represent the greatest plant abiotic stresses in crops. Increasing plant tolerance against these environmental conditions must be a key strategy in the development of future agriculture. The genus of Trichoderma filament fungi includes several species widely used as biocontrol agents for plant diseases but also some with the ability to increase plant tolerance against abiotic stresses. In this sense, using the species T. parareesei and T. harzianum, we have verified the differences between the two after their application in rapeseed (Brassica napus) root inoculation, with T. parareesei being a more efficient alternative to increase rapeseed productivity under drought or salinity conditions. In addition, we have determined the role that T. parareesei chorismate mutase plays in its ability to promote tolerance to salinity and drought in plants by increasing the expression of genes related to the hormonal pathways of abscisic acid (ABA) under drought stress, and ethylene (ET) under salt stress.

Plants, fungi, and bacteria synthesize the aromatic amino acids: L-phenylalanine, L-tyrosine, and L-tryptophan. Chorismate mutase catalyzes the branch point reaction of phenylalanine and tyrosine biosynthesis to generate prephenate. In Arabidopsis thaliana, there are two plastid-localized chorismate mutases that are allosterically regulated (AtCM1 and AtCM3) and one cytosolic isoform (AtCM2) that is unregulated. Previous analysis of plant chorismate mutases suggested that the enzymes from early plants (i.e. bryophytes/moss, lycophytes, and basal angiosperms) formed a clade distinct from the isoforms found in flowering plants; however, no biochemical information on these enzymes is available. To understand the evolution of allosteric regulation in plant chorismate mutases, we analyzed a basal lineage of plant enzymes homologous to AtCM1 based on sequence similarity. The chorismate mutases from the moss/bryophyte Physcomitrella patens (PpCM1 and PpCM2), the lycophyte Selaginella moellendorffii (SmCM), and the basal angiosperm Amborella trichopoda (AmtCM1 and AmtCM2) were characterized biochemically. Tryptophan was a positive effector for each of the five enzymes examined. Histidine was a weak positive effector for PpCM1 and AmtCM1. Neither tyrosine nor phenylalanine altered the activity of SmCM; however, tyrosine was a negative regulator of the other four enzymes. Phenylalanine down-regulates both moss enzymes and AmtCM2. The 2.0 Å X-ray crystal structure of PpCM1 in complex with the tryptophan identified the allosteric effector site and reveals structural differences between the R- (more active) and T-state (less active) forms of plant chorismate mutases. Molecular insight into the basal plant chorismate mutases guides our understanding of the evolution of allosteric regulation in these enzymes. [ABSTRACT FROM AUTHOR]

Chorismate mutase is a well-known model enzyme, catalyzing the Claisen rearrangement of chorismate to prephenate. Recent high-resolution crystal structures along the reaction coordinate of this enzyme enabled computational analyses at unprecedented detail. Using quantum chemical simulations, we investigated how the catalytic reaction mechanism is affected by electrostatic and hydrogen-bond interactions. Our calculations showed that the transition state (TS) was mainly stabilized electrostatically, with Arg90 playing the leading role. The effect was augmented by selective hydrogenbond formation to the TS in the wild-type enzyme, facilitated by a smallscale local induced fit. We further identified a previously underappreciated water molecule, which separates the negative charges during the reaction. The analysis includes the wild-type enzyme and a non-natural enzyme variant, where the catalytic arginine was replaced with an isosteric citrulline residue. [ABSTRACT FROM AUTHOR]

We have engineered the protein interface of the Escherichia coli chorismate mutase (EcCM) homodimer to be dependent on incorporation of a noncanonical amino acid (ncAA) at residue 72. The large hydrophobic amino acid p-benzoyl phenylalanine (pBzF) was substituted for Tyr72, which led to a catalytically inactive protein. A library of five residues (Leu25', Arg29', Leu76, Ile80' and Asp83') surrounding pBzF72 was generated and subjected to a growth based selection in a chorismate mutase deficient strain. An EcCM variant (Phe25', pBzF72, Thr76, Gly80' and Tyr83') forms a stable homodimer, has catalytic activity similar to the wild type enzyme, and unfolds with a T m of 53°C. The X-ray crystal structure reveals a pi-pi stacking and hydrogen bonding interactions that stabilize the new protein interface. The strategy described here should be useful for generating organisms that are dependent on the presence of a ncAA for growth. [ABSTRACT FROM AUTHOR]

Plant anthocyanin biosynthesis is well understood, but the regulatory mechanism in purple foliage tea remains unclear. Using isobaric tag for relative and absolute quantification (iTRAQ), 815 differential proteins were identified in the leaves of Zijuan tea, among which 20 were associated with the regulation of anthocyanin metabolism. We found that the abundances of anthocyanin synthesis-related enzymes such as chalcone synthase, chalcone isomerase, dihydroflavonol 4-reductase and anthocyanin synthetase, as well as anthocyanin accumulation-related UDP-glucosyl transferase and ATP-binding cassette (ABC) transporters in the purple leaves were all significantly higher than those in the green leaves. The abundances of the transcription factors bHLH and HY5, regulating anthocyanin biosynthesis at transcriptional level were also obviously higher in purple leaves than those in green leaves. In addition, bifunctional 3-dehydroquinate dehydratase and chorismate mutase in purple leaves were distinctly higher in abundance compared to green leaves, which provided sufficient phenylalanine substrate for anthocyanin synthesis. Furthermore, lignin synthesis was found to be reduced due to the lower abundances of cinnamoyl-CoA reductase 1, peroxidase 15 and laccase-6, which resulted in increase of intermediates flow into anthocyanin synthesis pathway. The physiological data were consistent with proteomic results. These four aspects of biosynthetic regulation contribute to anthocyanin accumulation in purple leaves of Zijuan tea. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis chorismate mutase ( Mtb CM) catalyzes the rearrangement of chorismate to prephenate in the shikimate biosynthetic pathway to form the essential amino acids, phenylalanine and tyrosine. Two genes encoding chorismate mutase have been identified in Mtb . The secretory form,∗ Mtb CM (encoded by Rv1885c) is assumed to play a key role in pathogenesis of tuberculosis. Also, the inhibition of Mtb CM may hinder the supply of nutrients to the organism. Indeed, the existence of chorismate mutase (CM) in bacteria, fungi and higher plants but not in human and low sequence homology among known CM makes it an interesting target for the discovery of anti-tubercular agents. The present article mainly focuses on the recent developments in the structure, function and inhibition of Mtb CM. The understanding of various aspects of Mtb CM as presented in the current article may facilitate the design and subsequent chemical synthesis of new inhibitors against ∗ Mtb CM, that could lead to the discovery and development of novel and potent anti-tubercular agents in future. [ABSTRACT FROM AUTHOR]

Biosynthesis of l -tyrosine ( l -Tyr) is directed by the interplay of two enzymes. Chorismate mutase (CM) catalyzes the rearrangement of chorismate to prephenate, which is then converted to hydroxyphenylpyruvate by prephenate dehydrogenase (PD). This work reports the first characterization of the independently expressed PD domain of bifunctional CM-PD from the crenarchaeon Ignicoccus hospitalis and the first functional studies of both full-length CM-PD and the PD domain from the bacterium Haemophilus influenzae . All proteins were hexa-histidine tagged, expressed in Escherichia coli and purified. Expression and purification of I. hospitalis CM-PD generated a degradation product identified as a PD fragment lacking the protein's first 80 residues, Δ80CM-PD. A comparable stable PD domain could also be generated by limited tryptic digestion of this bifunctional enzyme. Thus, Δ80CM-PD constructs were prepared in both organisms. CM-PD and Δ80CM-PD from both organisms were dimeric and displayed the predicted enzymatic activities and thermal stabilities in accord with their hyperthermophilic and mesophilic origins. In contrast with H. influenzae PD activity which was NAD + -specific and displayed > 75% inhibition with 50 μM l -Tyr, I. hospitalis PD demonstrated dual cofactor specificity with a preference for NADP + and an insensitivity to l -Tyr. These properties are consistent with a model of the I. hospitalis PD domain based on the previously reported structure of the H. influenzae homolog. Our results highlight the similarities and differences between the archaeal and bacterial TyrA proteins and reveal that the PD activity of both prokaryotes can be successfully mapped to a functionally independent unit. [ABSTRACT FROM AUTHOR]

The Claisen rearrangement of chorismate to prephenate is mapped across the entire reaction pathway using the fragment molecular orbital (FMO) method. Three basis sets (6-31G(d), cc-pVDZ, and pcseg-1) are studied to provide guidance toward obtaining high accuracy with the FMO method on such systems. Using a fragmentation scheme of one residue per fragment, the FMO method using the 6-31G(d) basis set and second-order Møller-Plesset perturbation theory (MP2) with the hybrid orbital projection fragmentation scheme provides the most reliable results across the entire reaction pathway. Calculations using the multilayer FMO method are performed and shown to be in agreement with single-layer calculations in all cases with differences of less than one kilocalorie per mole for all tested basis set combinations along the entire reaction path. The use of restricted Hartree-Fock for the lower-level layer and MP2 for the higher-level layer gives the most consistent results when using the same basis set for both layers. Pair interaction energy decomposition analysis calculations confirm that electrostatic interactions are the predominant force between three key arginine residues and chorismate and that dispersion and charge transfer interactions in the binding pocket also play a role in the local chemistry of the reaction. [ABSTRACT FROM AUTHOR]

Amino acids (AA) are essential molecules for plant physiology, acting as precursor molecules for proteins and other organic compounds. Chloroplasts play a vital role in AA metabolism, yet little is known about the impact on AA metabolism of albino plants' lack of chloroplasts. In this study, we conducted a quantitative proteome analysis on albino and variegated somaclonal variants of Agave angustifolia Haw. to investigate metabolic alterations in chloroplast-deficient plants, with a focus on AA metabolic pathways. We identified 82 enzymes involved in AA metabolism, with 32 showing differential accumulation between the somaclonal variants. Aa CM, Aa ALS, Aa BCAT, Aa IPMS1, Aa SHMT, Aa AST, Aa CGS, and Aa MS enzymes were particularly relevant in chloroplast-deficient Agave plantlets. Both variegated and albino phenotypes exhibited excessive synthesis of AA typically associated with chloroplasts (aromatic AAs, BCAAs, Asp, Lys, Pro and Met). Consistent trends were observed for Aa BCAT and Aa CM at mRNA and protein levels in albino plantlets. These findings highlight the critical activation and reprogramming of AA metabolic pathways in plants lacking chloroplasts. This study contributes to unraveling the intricate relationship between AA metabolism and chloroplast absence, offering insights into survival mechanisms of albino plants. • Proteome analysis is useful for examining amino acid metabolic pathways in plants with chloroplast abnormalities. • Proteomic data shows altered amino acid metabolism enzymes in albino Agave plantlets. • Albino Agave plantlets over-accumulate chloroplast-synthesized amino acids, suggesting metabolic imbalance. • Albino plantlets adapt to environment by altering amino acid metabolism enzymes. [ABSTRACT FROM AUTHOR]

• Sonochemical synthesis of 1,2,4-benzothiadiazine-1,1-dioxides is described. • Synthesis involved sequential use of Wang-OSO 3 H catalyst followed by NaHSO 3. • Three compounds e.g. 3c, 3d and 3e interacted well with Mtb CM in silico. • They also showed encouraging inhibition of Mtb CM in vitro. The sonochemical synthesis, in silico assessment and in vitro Mtb CM inhibitory activities of a series of 1,2,4-benzothiadiazine-1,1-dioxide derivatives are described. These compounds were synthesized via a one-pot two-step sonochemical method involving the Wang-OSO 3 H catalyzed reaction of 2-aminobenzenesulfonamide with aldehydes followed by treatment with NaHSO 3 in the same pot. The reaction proceeded at room temp in pure water affording the desired products in good yields. The use of heterogeneous catalyst, common oxidant, water as a solvent and ultrasound as the source of green energy in addition to the mild conditions, shorter reaction time and simple operational procedure are the key features of this methodology. In silico studies suggested that most of the synthesized compounds interacted with the external surface pockets of the Mtb CM (PDB: 2FP2) active site cavity. Indeed, a curved loop site was noted where these compounds were binding and aligned. Three compounds e.g. 3c, 3d and 3e interacted well with Mtb CM via the -NHSO 2 - moiety of their 2 H -benzo[ e ][1,2,4]thiadiazine-1,1-dioxide ring showing a common H-bond with SER70. They also showed good (55–63%) inhibition of Mtb CM in vitro when tested at 10 µM. According to the SAR study a 4-substututed phenyl ring was preferred over a 3- or 2-substutited phenyl moiety at the C-2 position and the 4-MeOC 6 H 4 substituent at this position was most effective in terms of activity. On the other hand, mediocre to low activity was observed when a heteroaryl ring or the bulky 2-naphthyl moiety was present at the C-2 position. Based on in silico and in vitro studies along with the ADME predictions the 1,2,4-benzothiadiazine-1,1-dioxide derivatives 3c, 3d and 3e emerged as pre-hits for further pharmacological evaluations. [Display omitted] We describe the one-pot sonochemical synthesis, in silico assessment and in vitro Mtb CM inhibitory activities of a series of 1,2,4-benzothiadiazine-1,1-dioxide derivatives. [ABSTRACT FROM AUTHOR]