Tryptophan production by catalysis of a putative tryptophan synthase protein.

Essential amino acid, tryptophan which intake from food plays a critical role in numerous metabolic functions, exhibiting extensive biological functions and applications. Tryptophan is beneficial for the food sector by enhancing nutritional content and promoting the development of functional foods. A putative gene encoding tryptophan synthase was the first identified in Sphingobacterium soilsilvae Em02, a cellulosic bacterium making it inherently more environmentally friendly. The gene was cloned and expressed in exogenous host Escherichia coli, to elucidate its function. The recombinant tryptophan synthase with a molecular weight 42 KDa was expressed in soluble component. The enzymatic activity to tryptophan synthase in vivo was assessed using indole and L-serine and purified tryptophan synthase. The optimum enzymatic activity for tryptophan synthase was recorded at 50 ºC and pH 7.0, which was improved in the presence of metal ions Mg2+, Sr2+ and Mn2+, whereas Cu2+, Zn2+ and Co2+ proved to be inhibitory. Using site-directed mutagenesis, the consensus pattern HK-S-[GGGSN]-E-S in the tryptophan synthase was demonstrated with K100Q, S202A, G246A, E361A and S385A as the active sites. Tryptophan synthase has been demonstrated to possess the defining characteristics of the ß-subunits. The tryptophan synthase may eventually be useful for tryptophan production on a larger scale. Its diverse applications highlight the potential for improving both the quality and health benefits of food products, making it an essential component in advancing food science and technology.

A combinatorially complete epistatic fitness landscape in an enzyme active site.

Protein engineering often targets binding pockets or active sites which are enriched in epistasis-nonadditive interactions between amino acid substitutions-and where the combined effects of multiple single substitutions are difficult to predict. Few existing sequence-fitness datasets capture epistasis at large scale, especially for enzyme catalysis, limiting the development and assessment of model-guided enzyme engineering approaches. We present here a combinatorially complete, 160,000-variant fitness landscape across four residues in the active site of an enzyme. Assaying the native reaction of a thermostable ß-subunit of tryptophan synthase (TrpB) in a nonnative environment yielded a landscape characterized by significant epistasis and many local optima. These effects prevent simulated directed evolution approaches from efficiently reaching the global optimum. There is nonetheless wide variability in the effectiveness of different directed evolution approaches, which together provide experimental benchmarks for computational and machine learning workflows. The most-fit TrpB variants contain a substitution that is nearly absent in natural TrpB sequences-a result that conservation-based predictions would not capture. Thus, although fitness prediction using evolutionary data can enrich in more-active variants, these approaches struggle to identify and differentiate among the most-active variants, even for this near-native function. Overall, this work presents a large-scale testing ground for model-guided enzyme engineering and suggests that efficient navigation of epistatic fitness landscapes can be improved by advances in both machine learning and physical modeling.

Fungi Tryptophan Synthases: What Is the Role of the Linker Connecting the α and ß Structural Domains in Hemileia vastatrix TRPS? A Molecular Dynamics Investigation.

Tryptophan synthase (TRPS) is a complex enzyme responsible for tryptophan biosynthesis. It occurs in bacteria, plants, and fungi as an αßßα heterotetramer. Although encoded by independent genes in bacteria and plants, in fungi, TRPS is generated by a single gene that concurrently expresses the α and ß entities, which are linked by an elongated peculiar segment. We conducted 1 µs all-atom molecular dynamics simulations on Hemileia vastatrix TRPS to address two questions: (i) the role of the linker segment and (ii) the comparative mode of action. Since there is not an experimental structure, we started our simulations with homology modeling. Based on the results, it seems that TRPS makes use of an already-existing tunnel that can spontaneously move the indole moiety from the α catalytic pocket to the ß one. Such behavior was completely disrupted in the simulation without the linker. In light of these results and the αß dimer's low stability, the full-working TRPS single genes might be the result of a particular evolution. Considering the significant losses that Hemileia vastatrix causes to coffee plantations, our next course of action will be to use the TRPS to look for substances that can block tryptophan production and therefore control the disease.

One-Pot Synthesis of Useful S-Substituted-l-cysteine Sulfoxides Using Genetically Engineered Escherichia coli.

S-Substituted-l-cysteine sulfoxides are valuable compounds that are contained in plants. Particularly, (+)-alliin and its degraded products have gained significant attention because of their human health benefits. However, (+)-alliin production has been limited to extraction from plants and chemical synthesis; both methods have drawbacks in terms of stability and safety. Here, we proposed the enzymatic cascade reaction for synthesizing (+)-alliin from readily available substrates. To achieve a one-pot (+)-alliin production, we constructed Escherichia coli coexpressing the genes encoding tryptophan synthase from Aeromonas hydrophila ssp. hydrophila NBRC 3820 and l-isoleucine hydroxylase from Bacillus thuringiensis 2e2 for the biocatalyst. Deletion of tryptophanase gene in E. coli increased the yield about 2-fold. Under optimized conditions, (+)-alliin accumulation reached 110 mM, which is the highest productivity thus far. Moreover, natural and unnatural S-substituted-l-cysteine sulfoxides were synthesized by applying various thiols to the cascade reaction. These results indicate that the developed bioprocess would enable the supply of diverse S-substituted-l-cysteine sulfoxides.

Prenylation of dimeric cyclo-L-Trp-L-Trp by the promiscuous cyclo-L-Trp-L-Ala prenyltransferase EchPT1.

Prenyltransferases (PTs) from the dimethylallyl tryptophan synthase (DMATS) superfamily are known as efficient biocatalysts and mainly catalyze regioselective Friedel-Crafts alkylation of tryptophan and tryptophan-containing cyclodipeptides (CDPs). They can also use other unnatural aromatic compounds as substrates and play therefore a pivotal role in increasing structural diversity and biological activities of a broad range of natural and unnatural products. In recent years, several prenylated dimeric CDPs have been identified with wide range of bioactivities. In this study, we demonstrate the production of prenylated dimeric CDPs by chemoenzymatic synthesis with a known promiscuous enzyme EchPT1, which uses cyclo-L-Trp-L-Ala as natural substrate for reverse C2-prenylation. High product yields were achieved with EchPT1 for C3-N1' and C3-C3' linked dimers of cyclo-L-Trp-L-Trp. Isolation and structural elucidation confirmed the product structures to be reversely C19/C19'-mono- and diprenylated cyclo-L-Trp-L-Trp dimers. Our study provides an additional example for increasing structural diversity by prenylation of complex substrates with known biosynthetic enzymes. KEY POINTS: • Chemoenzymatic synthesis of prenylated cyclo-L-Trp-L-Trp dimers • Same prenylation pattern and position for cyclodipeptides and their dimers. • Indole prenyltransferases such as EchPT1 can be widely used as biocatalysts.

"Biosynthesis of psilocybin and its nonnatural derivatives by a promiscuous psilocybin synthesis pathway in Escherichia coli".

Traditional psychedelics are undergoing a transformation from recreational drugs, to promising pharmaceutical drug candidates with the potential to provide an alternative treatment option for individuals struggling with mental illness. Sustainable and economic production methods are thus needed to facilitate enhanced study of these drug candidates to support future clinical efforts. Here, we expand upon current bacterial psilocybin biosynthesis by incorporating the cytochrome P450 monooxygenase, PsiH, to enable the de novo production of psilocybin as well as the biosynthesis of 13 psilocybin derivatives. The substrate promiscuity of the psilocybin biosynthesis pathway was comprehensively probed by using a library of 49 single-substituted indole derivatives, providing biophysical insights to this understudied metabolic pathway and opening the door to the in vivo biological synthesis of a library of previously unstudied pharmaceutical drug candidates.

Tryptophan-synthesizing bacteria enhance colonic motility.

BACKGROUND: An emerging strategy to treat symptoms of gastrointestinal (GI) dysmotility utilizes the administration of isolated bacteria. However, the underlying mechanisms of action of these bacterial agents are not well established. Here, we elucidate a novel approach to promote intestinal motility by exploiting the biochemical capability of specific bacteria to produce the serotonin (5-HT) precursor, tryptophan (Trp). METHODS: Mice were treated daily for 1 week by oral gavage of Bacillus (B.) subtilis (R0179), heat-inactivated R0179, or a tryptophan synthase-null strain of B. subtilis (1A2). Tissue levels of Trp, 5-HT, and 5-hydroxyindoleacetic acid (5-HIAA) were measured and changes in motility were evaluated. KEY RESULTS: Mice treated with B. subtilis R0179 exhibited greater colonic tissue levels of Trp and the 5-HT breakdown product, 5-HIAA, compared to vehicle-treated mice. Furthermore, B. subtilis treatment accelerated colonic motility in both healthy mice as well as in a mouse model of constipation. These effects were not observed with heat-inactivated R0179 or the live 1A2 strain that does not express tryptophan synthase. Lastly, we found that the prokinetic effects of B. subtilis R0179 were blocked by coadministration of a 5-HT4 receptor (5-HT4 R) antagonist and were absent in 5-HT4 R knockout mice. CONCLUSIONS AND INFERENCES: Taken together, these data demonstrate that intestinal motility can be augmented by treatment with bacteria that synthesize Trp, possibly through increased 5-HT signaling and/or actions of Trp metabolites, and involvement of the 5-HT4 R. Our findings provide mechanistic insight into a transient and predictable bacterial strategy to promote GI motility.

Structure-guided Mutagenesis Reveals the Catalytic Residue that Controls the Regiospecificity of C6-Indole Prenyltransferases.

Indole is a significant structural moiety and functionalization of the C-H bond in indole-containing molecules expands their chemical space, and modifies their properties and/or activities. Indole prenyltransferases (IPTs) catalyze the direct regiospecific installation of prenyl, C5 carbon units, on indole-derived compounds. IPTs have shown relaxed substrate flexibility enabling them to be used as tools for indole functionalization. However, the mechanism by which certain IPTs target a specific carbon position is not fully understood. Herein, we use structure-guided site-directed mutagenesis, in vitro enzymatic reactions, kinetics and structural-elucidation of analogs to verify the key catalytic residues that control the regiospecificity of all characterized regiospecific C6 IPTs. Our results also demonstrate that substitution of PriB_His312 to Tyr leads to the synthesis of analogs prenylated at different positions than C6. This work contributes to understanding of how certain IPTs can access a challenging position in indole-derived compounds.

Molecular cloning and functional characterization of BcTSA in the biosynthesis of indole alkaloids in Baphicacanthus cusia.

Baphicacanthus cusia (Nees) Bremek (B. cusia) is an essential traditional Chinese herb that is commonly used to treat colds, fever, and influenza. Indole alkaloids, such as indigo and indirubin, are the primary active constituents of B. cusia. The indole-producing reaction is crucial for regulating the flow of indole alkaloids metabolites along the pathways and coordinating primary and secondary product biosynthesis in plants. The tryptophan synthase alpha-subunit (TSA) can catalyse a process that produces indole, which is free to enter secondary metabolite pathways; however, the underlying potential mechanism of regulating indigo alkaloids synthesis remains unknown. Here, a BcTSA was cloned from the transcriptome of B. cusia. The BcTSA has a significant degree of similarity with other plant TSAs according to bioinformatics and phylogenetic analyses. Quantitative real-time PCR (RT-qPCR) research showed that BcTSA was dramatically enhanced in response to treatment with methyl jasmonate (MeJA), salicylic acid (SA), and abscisic acid (ABA), and was predominantly expressed in the stems as opposed to the leaves and rhizomes. Subcellular localization revealed that BcTSA is localized in chloroplasts, which is compatible with the fact that the conversion of indole-3-glycerol phosphate (IGP) to indole occurs in chloroplasts. The complementation assay results showed that BcTSA was functional, demonstrating that it was capable of catalyzing the conversion of IGP to indole. BcTSA was shown to stimulate the manufacture of indigo alkaloids including isatin, indigo, and indirubin when the gene was overexpressed in the hairy roots of Isatis indigotica. In conclusion, our research provides novel perspectives that might be applied to manipulating the indole alkaloid composition of B. cusia.

Comparative Coexpression Analysis of Indole Synthase and Tryptophan Synthase A Reveals the Independent Production of Auxin via the Cytosolic Free Indole.

Indole synthase (INS), a homologous cytosolic enzyme of the plastidal tryptophan synthase A (TSA), has been reported as the first enzyme in the tryptophan-independent pathway of auxin synthesis. This suggestion was challenged as INS or its free indole product may interact with tryptophan synthase B (TSB) and, therefore, with the tryptophan-dependent pathway. Thus, the main aim of this research was to find out whether INS is involved in the tryptophan-dependent or independent pathway. The gene coexpression approach is widely recognized as an efficient tool to uncover functionally related genes. Coexpression data presented here were supported by both RNAseq and microarray platforms and, hence, considered reliable. Coexpression meta-analyses of Arabidopsis genome was implemented to compare between the coexpression of TSA and INS with all genes involved in the production of tryptophan via the chorismate pathway. Tryptophan synthase A was found to be coexpressed strongly with TSB1/2, anthranilate synthase A1/B1, phosphoribosyl anthranilate transferase1, as well as indole-3-glycerol phosphate synthase1. However, INS was not found to be coexpressed with any target genes suggesting that it may exclusively and independently be involved in the tryptophan-independent pathway. Additionally, annotation of examined genes as ubiquitous or differentially expressed were described and subunits-encoded genes available for the assembly of tryptophan and anthranilate synthase complex were suggested. The most probable TSB subunits expected to interact with TSA is TSB1 then TSB2. Whereas TSB3 is only used under limited hormone conditions to assemble tryptophan synthase complex, putative TSB4 is not expected to be involved in the plastidial synthesis of tryptophan in Arabidopsis.

Tryptophan synthase ß subunit 1 affects stomatal phenotypes in Arabidopsis thaliana.

Stomata open in response to several environmental stimuli, such as light and low CO2. Plasma membrane (PM) H+-ATPase in guard cells plays a pivotal role for light-induced stomatal opening. In contrast, stomata close in response to the dark or plant hormone abscisic acid (ABA). However, molecular mechanisms of stomatal movements remain unclear. To elucidate the molecular mechanism of stomatal movements, we performed a genetic screen based on stomatal aperture-dependent weight decrease of detached leaves from EMS-treated Arabidopsis thaliana and isolated a rapid transpiration in detached leaves 2 (rtl2). The rtl2 mutant showed constitutive open-stomata phenotype with lower leaf temperature. ABA had no effect on stomatal aperture in rtl2. The rtl2 mutant also showed increased stomatal density, severe dwarf phenotype with pale green leaves and dark veins. Map-based analysis of the RTL2 locus revealed that the rtl2 mutant possesses a single nucleotide substitution, which induces amino acid substitution Gly162 to Glu in the tryptophan synthase ß subunit 1 (TSB1). The TSB1 encodes an enzyme in tryptophan (Trp) biosynthetic pathway. Amount of TSB1 protein was drastically reduced in rtl2 mutant. A different allele of tsb1 mutant (tsb1-1) also showed constitutive open-stomata phenotype with reduced TSB1 protein as in rtl2. Analyses of test-crossed plants of rtl2 and tsb1-1 showed open-stomata and dwarf phenotypes. These results indicate that a responsible gene for rtl2 is TSB1. We further investigated stomatal phenotype in mutants from Trp biosynthetic pathway, such as wei2-1 wei7-1, trp3-1, and tsb2-1. The trp3-1 mutant showed significant wider stomatal aperture as well as tsb1-1. Trp biosynthetic pathway closely relates to auxin biosynthesis. Then, we investigated auxin responsible genes and found that an expression of AUR3 was up in rtl2. In contrast, auxin had no effect on stomatal aperture in Arabidopsis and the phosphorylation status of PM H+-ATPase in guard cell protoplasts from Vicia faba. In addition, auxin antagonist had no effect on stomatal aperture. Interestingly, tsb1-1 grown under hydroponic culture system showed normal stomatal aperture by exogenously application of Trp. These results suggest that open stomata phenotype in tsb1-1 is due to Trp deficiency but not auxin.

Allosteric regulation of substrate channeling: Salmonella typhimurium tryptophan synthase.

The regulation of the synthesis of L-tryptophan (L-Trp) in enteric bacteria begins at the level of gene expression where the cellular concentration of L-Trp tightly controls expression of the five enzymes of the Trp operon responsible for the synthesis of L-Trp. Two of these enzymes, trpA and trpB, form an αßßα bienzyme complex, designated as tryptophan synthase (TS). TS carries out the last two enzymatic processes comprising the synthesis of L-Trp. The TS α-subunits catalyze the cleavage of 3-indole D-glyceraldehyde 3'-phosphate to indole and D-glyceraldehyde 3-phosphate; the pyridoxal phosphate-requiring ß-subunits catalyze a nine-step reaction sequence to replace the L-Ser hydroxyl by indole giving L-Trp and a water molecule. Within αß dimeric units of the αßßα bienzyme complex, the common intermediate indole is channeled from the α site to the ß site via an interconnecting 25 Å-long tunnel. The TS system provides an unusual example of allosteric control wherein the structures of the nine different covalent intermediates along the ß-reaction catalytic path and substrate binding to the α-site provide the allosteric triggers for switching the αßßα system between the open (T) and closed (R) allosteric states. This triggering provides a linkage that couples the allosteric conformational coordinate to the covalent chemical reaction coordinates at the α- and ß-sites. This coupling drives the α- and ß-sites between T and R conformations to achieve regulation of substrate binding and/or product release, modulation of the α- and ß-site catalytic activities, prevention of indole escape from the confines of the active sites and the interconnecting tunnel, and synchronization of the α- and ß-site catalytic activities. Here we review recent advances in the understanding of the relationships between structure, function, and allosteric regulation of the complex found in Salmonella typhimurium.

Estimating conformational heterogeneity of tryptophan synthase with a template-based Alphafold2 approach.

The three-dimensional structure of the enzymes provides very relevant information on the arrangement of the catalytic machinery and structural elements gating the active site pocket. The recent success of the neural network Alphafold2 in predicting the folded structure of proteins from the primary sequence with high levels of accuracy has revolutionized the protein design field. However, the application of Alphafold2 for understanding and engineering function directly from the obtained single static picture is not straightforward. Indeed, understanding enzymatic function requires the exploration of the ensemble of thermally accessible conformations that enzymes adopt in solution. In the present study, we evaluate the potential of Alphafold2 in assessing the effect of the mutations on the conformational landscape of the beta subunit of tryptophan synthase (TrpB). Specifically, we develop a template-based Alphafold2 approach for estimating the conformational heterogeneity of several TrpB enzymes, which is needed for enhanced stand-alone activity. Our results show the potential of Alphafold2, especially if combined with molecular dynamics simulations, for elucidating the changes induced by mutation in the conformational landscapes at a rather reduced computational cost, thus revealing its plausible application in computational enzyme design.

Evidence from Co-expression Analysis for the Involvement of Amidase and INS in the Tryptophan-Independent Pathway of IAA Synthesis in Arabidopsis.

The reverse genetic approach has uncovered indole synthase (INS) as the first enzyme in the tryptophan (trp)-independent pathway of IAA synthesis. The importance of INS was reevaluated suggesting it may interact with tryptophan synthase B (TSB) and therefore involved in the trp-dependent pathway. Thus, the main aim of this study was to clarify the route of INS through the analysis of Arabidopsis genome. Analysis of the top 2000 co-expression gene lists in general and specific conditions shows that TSA is strongly positively co-expressed with TSB in general, hormone, and abiotic conditions with mutual ranks of 89, 38, and 180 respectively. Moreover, TSA is positively correlated with TSB (0.291). However, INS was not found in any of these coexpressed gene lists and negatively correlated with TSB (- 0.046) suggesting unambiguously that these two routes are separately and independently operated. So far, the remaining steps in the INS pathway have remained elusive. Among all enzymes reported to have a role in IAA synthesis, amidase was found to strongly positively co-expressed with INS in general and light conditions with mutual ranks of 116 and 141 respectively. Additionally, amidase1 was found to positively correlate with INS (0.297) and negatively coexpressed with TSB concluding that amidase may exclusively involve in the trp-independent pathway.

Fermentative Indole Production via Bacterial Tryptophan Synthase Alpha Subunit and Plant Indole-3-Glycerol Phosphate Lyase Enzymes.

Indole is produced in nature by diverse organisms and exhibits a characteristic odor described as animal, fecal, and floral. In addition, it contributes to the flavor in foods, and it is applied in the fragrance and flavor industry. In nature, indole is synthesized either from tryptophan by bacterial tryptophanases (TNAs) or from indole-3-glycerol phosphate (IGP) by plant indole-3-glycerol phosphate lyases (IGLs). While it is widely accepted that the tryptophan synthase α-subunit (TSA) has intrinsically low IGL activity in the absence of the tryptophan synthase ß-subunit, in this study, we show that Corynebacterium glutamicum TSA functions as a bona fide IGL and can support fermentative indole production in strains providing IGP. By bioprospecting additional bacterial TSAs and plant IGLs that function as bona fide IGLs were identified. Capturing indole in an overlay enabled indole production to titers of about 0.7 g L-1 in fermentations using C. glutamicum strains expressing either the endogenous TSA gene or the IGL gene from wheat.

l-Tryptophan-starved cultivation enhances S-allyl-l-cysteine synthesis in various food-related microorganisms.

S-Allyl-l-cysteine (SAC) has received much interest due to its beneficial effects on human health. To satisfy the increasing demand for SAC, this study aims to develop a valuable culturing method for microbial screening synthesizing SAC from readily available materials. Although tryptophan synthase is a promising enzyme for SAC synthesis, its expression in microorganisms is strictly regulated by environmental l-tryptophan. Thus, we constructed a semisynthetic medium lacking l-tryptophan using casamino acids. This medium successfully enhanced the SAC-synthesizing activity of Lactococcus lactis ssp. cremoris NBRC 100676. In addition, microorganisms with high SAC-synthesizing activity were screened by the same medium. Food-related Klebsiella pneumoniae K-15 and Pantoea agglomerans P-3 were found to have a significantly increased SAC-synthesizing activity. The SAC-producing process established in this study is shorter in duration than the conventional garlic aging method. Furthermore, this study proposes a promising alternative strategy for producing food-grade SAC by microorganisms.

Direct and indirect mechanisms by which the gut microbiota influence host serotonin systems.

Mounting evidence highlights the pivotal role of enteric microbes as a dynamic interface with the host. Indeed, the gut microbiota, located in the lumen of the gastrointestinal (GI) tract, influence many essential physiological processes that are evident in both healthy and pathological states. A key signaling molecule throughout the body is serotonin (5-hydroxytryptamine; 5-HT), which acts in the GI tract to regulate numerous gut functions including intestinal motility and secretion. The gut microbiota can modulate host 5-HT systems both directly and indirectly. Direct actions of gut microbes, evidenced by studies using germ-free animals or antibiotic administration, alter the expression of key 5-HT-related genes to promote 5-HT biosynthesis. Indirectly, the gut microbiota produce numerous microbial metabolites, whose actions can influence host serotonergic systems in a variety of ways. This review summarizes the current knowledge regarding mechanisms by which gut bacteria act to regulate host 5-HT and 5-HT-mediated gut functions, as well as implications for 5-HT in the microbiota-gut-brain axis.

Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate.

NMR-assisted crystallography-the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry-holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5'-phosphate-dependent enzymes that catalyze ß-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate ß-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid-base catalytic residue ßLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate Cß and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.

Atomic-resolution chemical characterization of (2x)72-kDa tryptophan synthase via four- and five-dimensional 1H-detected solid-state NMR.

NMR chemical shifts provide detailed information on the chemical properties of molecules, thereby complementing structural data from techniques like X-ray crystallography and electron microscopy. Detailed analysis of protein NMR data, however, often hinges on comprehensive, site-specific assignment of backbone resonances, which becomes a bottleneck for molecular weights beyond 40 to 45 kDa. Here, we show that assignments for the (2x)72-kDa protein tryptophan synthase (665 amino acids per asymmetric unit) can be achieved via higher-dimensional, proton-detected, solid-state NMR using a single, 1-mg, uniformly labeled, microcrystalline sample. This framework grants access to atom-specific characterization of chemical properties and relaxation for the backbone and side chains, including those residues important for the catalytic turnover. Combined with first-principles calculations, the chemical shifts in the ß-subunit active site suggest a connection between active-site chemistry, the electrostatic environment, and catalytically important dynamics of the portal to the ß-subunit from solution.

Comparative analysis of secondary metabolite gene clusters in different strains of Magnaporthe oryzae.

Rice blast caused by Magnaporthe oryzae continues to be a major constraint in rice production worldwide. Rice is one of the staple crops in India and rice blast causes huge economic losses. Interestingly, the Indian subcontinent is the centre for origin and diversity of rice as well as the Magnaporthe species complex. Secondary metabolites are known to play important role in pathogenesis and M. oryzae has high potential of genes involved in secondary metabolism but, unfortunately most of them remain uncharacterized. In the present study, we analysed the draft genome assemblies of M. oryzae strains isolated from different parts of India, for putative secondary metabolite key gene (SMKG) clusters encoding polyketide synthases, non-ribosomal peptide synthetases, diterpene cyclases and dimethylallyl tryptophan synthase. Based on the complete genome sequence of 70-15 strain and its previous reports of identified SMKGs, we have identified the key genes for the interrogated strains. Expression analysis of these genes amongst different strains indicates how they have evolved depending on the host and environmental conditions. To our knowledge, this study is first of its kind where the secondary metabolism genes and their role in functional adaptation were studied across several strains of M. oryzae.