Tryptophan Synthase Research Articles

Inhibiting mycobacterial tryptophan synthase by targeting the inter-subunit interface

Drug discovery efforts against the pathogen Mycobacterium tuberculosis (Mtb) have been advanced through phenotypic screens of extensive compound libraries. Such a screen revealed sulfolane 1 and indoline-5-sulfonamides 2 and 3 as potent inhibitors of mycobacterial growth. Optimization in the sulfolane series led to compound 4, which has proven activity in an in vivo murine model of Mtb infection. Here we identify the target and mode of inhibition of these compounds based on whole genome sequencing of spontaneous resistant mutants, which identified mutations locating to the essential α- and β-subunits of tryptophan synthase. Over-expression studies confirmed tryptophan synthase as the biological target. Biochemical techniques probed the mechanism of inhibition, revealing the mutant enzyme complex incurs a fitness cost but does not prevent inhibitor binding. Mapping of the resistance conferring mutations onto a low-resolution crystal structure of Mtb tryptophan synthase showed they locate to the interface between the α- and β-subunits. The discovery of anti-tubercular agents inhibiting tryptophan synthase highlights the therapeutic potential of this enzyme and draws attention to the prospect of other amino acid biosynthetic pathways as future Mtb drug targets.

Drug development: Locking down metabolism.

An allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase—an enzyme that is nonessential for in vitro growth—has potent antimicrobial activity, revealing a potentially expanded target list for antimicrobials and greater chemical space for new inhibitors.

Possible new way to attack tuberculosis

The bacterium Mycobacterium tuberculosis claimed 1.8 million lives worldwide in 2015, according to the World Health Organization, making tuberculosis the deadliest infectious disease on Earth. Scientists led by Deborah T. Hung of Broad Institute report a small molecule, called BRD4592, that inhibits one of the bacterium’s key metabolic enzymes—tryptophan synthase (Nat. Chem. Biol. 2017, DOI: 10.1038/nchembio.2420). BRD4592 shuts down this enzyme, not by binding to its active sites, but by binding in the channel between the enzyme’s subunits. This allosteric binding prevents the enzyme from completing the cycle it uses to make tryptophan. It also blocks indole, an important intermediate en route to tryptophan, from shuttling between the enzyme’s two active sites. Hung’s team believes this is one of the most complicated allosteric inhibitors reported to date. The scientists found that mice metabolized BRD4592 too quickly to evaluate the compound’s efficacy in infected animals, but the team did determine that

A small-molecule allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase

New antibiotics with novel targets are greatly needed. Bacteria have numerous essential functions, but only a small fraction of such processes-primarily those involved in macromolecular synthesis-are inhibited by current drugs. Targeting metabolic enzymes has been the focus of recent interest, but effective inhibitors have been difficult to identify. We describe a synthetic azetidine derivative, BRD4592, that kills Mycobacterium tuberculosis (Mtb) through allosteric inhibition of tryptophan synthase (TrpAB), a previously untargeted, highly allosterically regulated enzyme. BRD4592 binds at the TrpAB α-β-subunit interface and affects multiple steps in the enzyme's overall reaction, resulting in inhibition not easily overcome by changes in metabolic environment. We show that TrpAB is required for the survival of Mtb and Mycobacterium marinum in vivo and that this requirement may be independent of an adaptive immune response. This work highlights the effectiveness of allosteric inhibition for targeting proteins that are naturally highly dynamic and that are essential in vivo, despite their apparent dispensability under in vitro conditions, and suggests a framework for the discovery of a next generation of allosteric inhibitors.

Structure of Mycobacterium tuberculosis tryptophan synthase: a model system for allosteric inhibition

Structure of <i>Mycobacterium tuberculosis</i> tryptophan synthase: a model system for allosteric inhibition

Structural insight into allosteric inhibition of Mycobacterium tuberculosis tryptophan synthase

Structural insight into allosteric inhibition of <i>Mycobacterium tuberculosis</i> tryptophan synthase

NMR crystallography in the enzyme active site of tryptophan synthase

NMR crystallography in the enzyme active site of tryptophan synthase

RETRACTED: Expression system for heterologous protein expression in the filamentous fungus Aspergillus unguis

Heterologous protein expression in filamentous fungi is advantageous, especially in the context of large scale production of high volume low value recombinant proteins. However, such systems are rare and not available in public domain. A novel filamentous fungus – Aspergillus unguis NII 08123 was used as host for developing a protein expression system. An expression cassette was assembled using A. nidulans glyceraldehyde 3 phosphate dehydrogenase promoter (Pgapd), tryptophan synthase transcription terminator (TtrpC) and hygromycin resistance gene (hph) as selection marker. The enhanced green fluorescent protein (GFP) gene from Aequorea victoria was used as the model test protein for the evaluation of the expression system. The genetic transformation of this novel fungus was optimized through electroporation. Use of heterologous signal peptides resulted in high levels of secreted expression. The fungal host-expression system combination was tested successfully for the expression of the recombinant therapeutic protein-human interferon beta (HuIFNβ).

Identifying the Genes Regulated by AtWRKY6 Using Comparative Transcript and Proteomic Analysis under Phosphorus Deficiency.

Phosphorus (P) is an important mineral nutrient for plant growth and development. Overexpressing AtWRKY6 (35S:WRKY6-9) was more sensitive and wrky6 (wrky6-1) was more resistant under low Pi conditions. To better understand the function of AtWRKY6 under low phosphate stress conditions, we applied two-dimensional gel electrophoresis (2-DE) to analyse differentially expressed proteins in the shoots and roots between wild type, 35S:WRKY6-9 and wrky6-1 after phosphorus deficiency treatment for three days. The results showed 88 differentially abundant protein spots, which were identified between the shoots and roots of 35S:WRKY6-9 and wrky6-1 plants. In addition, 59 differentially expressed proteins were identified in the leaves and roots of 35S:WRKY6-9 plants. After analysis, 9 genes with W-box elements in their promoter sequences were identified in the leaves, while 6 genes with W-box elements in their promoter sequences were identified in the roots. A total of 8 genes were identified as potential target genes according to the quantitative PCR (QPCR) and two dimension difference gel electrophoresis, (2D-DIGE) results, including ATP synthase, gln synthetase, nitrilase, 14-3-3 protein, carbonic anhydrases 2, and tryptophan synthase. These results provide important information concerning the AtWRKY6 regulation network and reveal potential vital target genes of AtWRKY6 under low phosphorus stress. two dimension difference gel electrophoresis, 2D-DIGE

Bio-suppression of turmeric rhizome rot disease and understanding the molecular basis of tripartite interaction among Curcuma longa, Pythium aphanidermatum and Pseudomonas fluorescens

Turmeric rhizome rot is a devastating disease posting the major threat to turmeric cultivation. Plant growth-promoting rhizobacteria (PGPR) Pseudomonas fluorescens strain FP7 was found to exhibit disease reduction and plant growth promotional activity in various crop plants in our previous studies. In this study, efforts were made to evaluate the efficacy of P. fluorescens (FP7) bioformulations against rhizome rot disease in turmeric plants. Among the FP7 bioformulations tested, a combination of rhizome dip and soil drench of FP7 liquid formulation recorded the minimum disease incidence under glasshouse (19.00%) and field conditions (10.18% and 13.29% in the trial I and trial II respectively). Furthermore, the 2D-PAGE analysis was performed to elucidate the molecular responses of tripartite interaction between host-pathogen-bioagent through protein profiling. A total of 12 differentially expressed proteins were identified and mass spectrometry (MS) analysis revealed that proteins such as tryptophan synthase beta subunit-like, phosphoglycerate kinase, cellulose synthase 6 isoform, ATP synthase subunit beta, cysteine-rich peptide, ribosomal protein S3, clathrin assembly protein and disease resistance protein RPP13-like were found to be differentially regulated. The differentially expressed proteins during tripartite interaction might be directly or indirectly involved in disease resistance in turmeric plants. This study provides an initial insight into the molecular mechanism of tripartite interaction between host-pathogen-bioagent in turmeric plants.

Systematic Dissociation Pathway Searches Guided by Principal Component Modes.

We introduce a novel method, Pathway Search guided by Internal Motions (PSIM), that efficiently finds molecular dissociation pathways of a ligand-receptor system with guidance from principal component (PC) modes obtained from molecular dynamics (MD) simulations. Modeling ligand-receptor dissociation pathways can provide insights into molecular recognition and has practical applications, including understanding kinetic mechanisms and barriers to binding/unbinding as well as design of drugs with desired kinetic properties. PSIM uses PC modes in multilayer internal coordinates to identify natural molecular motions that guide the search for conformational switches and unbinding pathways. The new multilayer internal coordinates overcome problems with Cartesian and classical internal coordinates that fail to smoothly present dihedral rotation or generate nonphysical distortions. We used HIV-1 protease, which has large-scale flap motions, as an example protein to demonstrate use of the multilayer internal coordinates. We provide examples of algorithms and implementation of PSIM with alanine dipeptide and chemical host-guest systems, 2-naphthyl ethanol-β-cyclodextrin and tetramethylammonium-cryptophane complexes. Tetramethylammonium-cryptophane has slow binding/unbinding kinetics. Its residence time, the length to dissociate tetramethylammonium from the host, is ∼14 s from experiments, and PSIM revealed 4 dissociation pathways in approximately 150 CPU h. We also searched the releasing pathways for the product glyceraldehyde-3-phosphate from tryptophan synthase, and one complete dissociation pathway was constructed after running multiple search iterations in approximately 300 CPU h. With guidance by internal PC modes from MD simulations, the PSIM method has advantages over simulation-based methods to search for dissociation pathways of molecular systems with slow noncovalent kinetic behavior.

Structural Insight into Allosteric Inhibition of Mycobacterium tuberculosis Tryptophan Synthase

The rise of antibiotic‐resistant pathogens requires redirecting drug discovery effort toward new cellular targets to provide new treatment options. Such alternative, yet unexplored opportunities exist in central metabolism, where many enzymes are only conditionally essential and thus their successful exploitation requires understanding of metabolic fluxes and pathway regulations under certain environmental conditions. In Mycobacterium tuberculosis, tryptophan biosynthesis represents one such promising pathway that could be targeted. In Mtb, the multistep L‐Trp synthesis involves several enzymes: TrpEG, TrpD, TrpC and TrpAB, which is validated as conditionally essential in vivo. The whole‐cell screening of a diversity‐oriented synthetic library identified a novel bactericidal azetidine derivative, BRD4592, that specifically inhibits TrpAB (tryptophan synthase). TrpAB is a heterotetrameric, PLP‐dependent complex catalyzing two last reactions in L‐Trp synthesis: the conversion of indole‐3‐glycerol phosphate to indole and glyceraldehyde 3‐phosphate (TrpA) followed by indole and L‐Ser condensation to yield L‐Trp (TrpB). During the catalytic cycle, indole is transferred from the TrpA to TrpB active site through 25 Å hydrophobic channel to react with the L‐Ser‐PLP adduct. The enzymatic activities and substrate channeling are coordinated by complex allosteric communication between the subunits. This regulation involves cycling between various conformational states, with both subunits adopting either an open (inactive) or closed (active) state. Detailed kinetic, biophysical and structural evidence reveals that BRD4592 is an allosteric, mixed‐type inhibitor, which binds specifically to a novel site within the intersubunit channel, at the TrpA‐TrpB interface. The molecule promotes closed, active state of the TrpB subunit and increases enzyme affinity for the L‐Ser substrate. BRD4592 acts on three levels: it limits flexibility of the interface potentially disrupting mutual activation of the subunits, prevents indole translocation, and increases product inhibition providing means to successfully inhibit both, TrpA and TrpB reactions. Our results indicate that allosteric binders may represent an effective tool to deactivate central metabolism enzymes, which often are allosterically regulated and might be predisposed for this type of inhibition.Support or Funding InformationThis work was supported by the Broad Institute Tuberculosis donor group, the Pershing Square Foundation, the Bill &amp; Melinda Gates Foundation under grant OPP1032518, the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contracts HHSN272200700058C and HHSN272201200026C and the U. S. Department of Energy, Office of Biological and Environmental Research, under contract DE‐AC02‐06CH11357. For this work, University of Chicago is a subgrantee of Structural Genomics Consortium under a Bill &amp; Melinda Gates Foundation grant for the Structure‐Guided Drug Discovery Coalition.

Precise through-space control of an abiotic electrophilic aromatic substitution reaction

Nature has evolved selective enzymes for the efficient biosynthesis of complex products. This exceptional ability stems from adapted enzymatic pockets, which geometrically constrain reactants and stabilize specific reactive intermediates by placing electron-donating/accepting residues nearby. Here we perform an abiotic electrophilic aromatic substitution reaction, which is directed precisely through space. Ester arms—positioned above the planes of aromatic rings—enable it to distinguish between nearly identical, neighbouring reactive positions. Quantum mechanical calculations show that, in two competing reaction pathways, both [C–H···O]–hydrogen bonding and electrophile preorganization by coordination to a carbonyl group likely play a role in controlling the reaction. These through-space-directed mechanisms are inspired by dimethylallyl tryptophan synthases, which direct biological electrophilic aromatic substitutions by preorganizing dimethylallyl cations and by stabilizing reactive intermediates with [C–H···N]–hydrogen bonding. Our results demonstrate how the third dimension above and underneath aromatic rings can be exploited to precisely control electrophilic aromatic substitutions.

CATALASE2 Coordinates SA-Mediated Repression of Both Auxin Accumulation and JA Biosynthesis in Plant Defenses

Plants defend against pathogen attack by modulating auxin signaling and activating the salicylic acid (SA) and jasmonic acid (JA) signaling pathways. SA and JA act antagonistically in resistance to specific pathogen types, yet how plants coordinate these phytohormones remains elusive. Here we report that biotrophic-pathogen-induced SA accumulation dampens both auxin and JA synthesis by inhibiting CATALASE2 (CAT2) activity in the model plant Arabidopsis. SA suppression of CAT2 results in increased H2O2 levels and subsequent sulfenylation of tryptophan synthetase β subunit 1, thus depleting the auxin biosynthetic precursor tryptophan. In addition, we find that CAT2 promotes JA biosynthesis by facilitating direct interaction of the JA biosynthetic enzymes ACX2 and ACX3, and thusSA repression of CAT2 inhibits JA accumulation. As such, the cat2-1 mutant exhibits increased resistance to biotrophic pathogens and increased susceptibility to necrotrophic pathogens. Our study illustrates how CAT2 coordinates SA repression of auxin accumulation and JA biosynthesis in plant defense.

An Engineered Tryptophan Synthase Opens New Enzymatic Pathways to β-Methyltryptophan and Derivatives.

β-Methyltryptophans (β-mTrp) are precursors in the biosynthesis of bioactive natural products and are used in the synthesis of peptidomimetic-based therapeutics. Currently β-mTrp is produced by inefficient multistep synthetic methods. Here we demonstrate how an engineered variant of tryptophan synthase from Salmonella (StTrpS) can catalyse the efficient condensation of l-threonine and various indoles to generate β-mTrp and derivatives in a single step. Although l-serine is the natural substrate for TrpS, targeted mutagenesis of the StTrpS active site provided a variant (βL166V) that can better accommodate l-Thr as a substrate. The condensation of l-Thr and indole proceeds with retention of configuration at both α- and β-positions to give (2S,3S)-β-mTrp. The integration of StTrpS (βL166V) with l-amino acid oxidase, halogenase enzymes and palladium chemocatalysts provides access to further d-configured and regioselectively halogenated or arylated β-mTrp derivatives.

Systematic characterization of artificial small RNA-mediated inhibition of Escherichia coli growth

ABSTRACTA new screening system for artificial small RNAs (sRNAs) that inhibit the growth of Escherichia coli was constructed. In this system, we used a plasmid library to express RNAs of ∼120 nucleotides, each with a random 30-nucleotide sequence that can recognize its target mRNA(s). After approximately 60,000 independent colonies were screened, several plasmids that inhibited bacterial growth were isolated. To understand the inhibitory mechanism, we focused on one sRNA, S-20, that exerted a strong inhibitory effect. A time-course analysis of the proteome of S-20-expressing E. coli and a bioinformatic analysis were used to identify potential S-20 target mRNAs, and suggested that S-20 binds the translation initiation sites of several mRNAs encoding enzymes such as peroxiredoxin (osmC), glycyl-tRNA synthetase α subunit (glyQ), uncharacterized protein ygiM, and tryptophan synthase β chain (trpB). An in vitro translation analysis of chimeric luciferase-encoding mRNAs, each containing a potential S-20 target sequence, indicated that the translation of these mRNAs was inhibited in the presence of S-20. A gel shift analysis combined with the analysis of a series of S-20 mutants suggested that S-20 targets multiple mRNAs that are responsible for inhibiting E. coli growth. These data also suggest that S-20 acts like an endogenous sRNA and that E. coli can utilize artificial sRNAs.

Stochastic thermodynamics of a chemical nanomachine: The channeling enzyme tryptophan synthase.

The enzyme tryptophan synthase is characterized by a complex pattern of allosteric interactions that regulate the catalytic activity of its two subunits and opening or closing of their ligand gates. As a single macromolecule, it implements 13 different reaction steps, with an intermediate product directly channeled from one subunit to another. Based on experimental data, a stochastic model for the operation of tryptophan synthase has been earlier constructed [D. Loutchko, D. Gonze, and A. S. Mikhailov, J. Phys. Chem. B 120, 2179 (2016)]. Here, this model is used to consider stochastic thermodynamics of such a chemical nanomachine. The Gibbs energy landscape of the internal molecular states is determined, the production of entropy and its flow within the enzyme are analyzed, and the information exchange between the subunits resulting from allosteric cross-regulations and channeling is discussed.

Protein stability and dynamics influenced by ligands in extremophilic complexes - a molecular dynamics investigation.

In this study, we explore the structural and dynamic adaptations of the Tryptophan synthase α-subunit in a ligand bound state in psychrophilic, mesophilic and hyperthermophilic organisms at different temperatures by MD simulations. We quantify the global and local fluctuations in the 40 ns time scale by analyzing the root mean square deviation/fluctuations. The distinct behavior of the active site and loop 6 is observed with the elevation of temperature. Protein stability relies more on electrostatic interactions, and these interactions might be responsible for the stability of varying temperature evolved proteins. The paper also focuses on the effect of temperature on protein dynamics and stability governed by the distinct behavior of the ligand associated with its retention, binding and dissociation over the course of time. The integration of principle component analysis and a free energy landscape was useful in identifying the conformational space accessible to ligand bound homologues and how the presence of the ligand alters the conformational and dynamic properties of the protein.

Non-Thermal Radio Frequency Stimulation Inhibits the Tryptophan Synthase Beta Subunit in the Algae &amp;lt;i&amp;gt;Chlamydomonas reinhardtii&amp;lt;/i&amp;gt;

To demonstrate the ability of the Nativis signal transduction technology (Butters et al. 2014) to modulate the expression of algae mRNA and protein, we tested if we can alter specific enzyme levels in Chlamydomonas reinhardtii. We inhibited the synthesis of the enzyme tryptophan synthase beta subunit (MAA7) by applying the signal derived from a published siRNA (Zhao et al. 2009). With lower levels of MAA7, Chlamydomonas reinhardtii can grow in the presence of the prodrug 5-Fluoroindole (5-FI), because less 5-Fluoroin-dole can be converted to the toxic 5-Fluoro-L-tryptophan (5-FT). We find a 24% (±5%) increase of growth with the signal versus no signal. To see if that effect was due to the reduction of the amount of mRNA encoding MAA7, we used Real-Time Quantitative PCR (RT-QPCR) to measure the levels of MAA7 mRNA. To normalize the MAA7 mRNA level, we compared them to the levels of a mRNA that is not affected by the signal (G protein beta subunit-like polypeptide, Cblp). Two conditions increase the effectiveness of the signal. One can either treat the cell cultures during the logarithmic growth phase (starting the cultures at density of 0.104 OD at 750 nm). Or one can treat the cultures at a later stage of the logarithmic growth, but treating them for a longer time (8.7% versus 3.5% of the culture time). Under these conditions we found around a 50% decrease in the mRNA levels for MAA7. Treating the cultures at the earlier growth phase or at a later growth phase is less effective, with only a 20% effect.

Tryptophan Synthase Uses an Atypical Mechanism To Achieve Substrate Specificity.

Tryptophan synthase (TrpS) catalyzes the final steps in the biosynthesis of l-tryptophan from l-serine (Ser) and indole-3-glycerol phosphate (IGP). We report that native TrpS can also catalyze a productive reaction with l-threonine (Thr), leading to (2S,3S)-β-methyltryptophan. Surprisingly, β-substitution occurs in vitro with a 3.4-fold higher catalytic efficiency for Ser over Thr using saturating indole, despite a >82000-fold preference for Ser in direct competition using IGP. Structural data identify a novel product binding site, and kinetic experiments clarify the atypical mechanism of specificity: Thr binds efficiently but decreases the affinity for indole and disrupts the allosteric signaling that regulates the catalytic cycle.