Catalytically impaired TrpA subunit of tryptophan synthase from Chlamydia trachomatis is an alloster
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Michalska, Karolina↗
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Microgravity crystallization of perdeuterated tryptophan synthase for neutron diffraction
Abstract Biologically active vitamin B 6 -derivative pyridoxal 5′-phosphate (PLP) is an essential cofactor in amino acid metabolic pathways. PLP-dependent enzymes catalyze a multitude of chemical reactions but, how reaction diversity of PLP-dependent enzymes is achieved is still not well understood. Such comprehension requires atomic-level structural studies of PLP-dependent enzymes. Neutron diffraction affords the ability to directly observe hydrogen positions and therefore assign protonation states to the PLP cofactor and key active site residues. The low fluxes of neutron beamlines require large crystals (≥0.5 mm 3 ). Tryptophan synthase (TS), a Fold Type II PLP-dependent enzyme, crystallizes in unit gravity with inclusions and high mosaicity, resulting in poor diffraction. Microgravity offers the opportunity to grow large, well-ordered crystals by reducing gravity-driven convection currents that impede crystal growth. We developed the Toledo Crystallization Box (TCB), a membrane-barrier capillary-dialysis device, to grow neutron diffraction-quality crystals of perdeuterated TS in microgravity. Here, we present the design of the TCB and its implementation on Center for Advancement of Science in Space (CASIS) supported International Space Station (ISS) Missions Protein Crystal Growth (PCG)-8 and PCG-15. The TCB demonstrated the ability to improve X-ray diffraction and mosaicity on PCG-8. In comparison to ground control crystals of the same size, microgravity-grown crystals from PCG-15 produced higher quality neutron diffraction data. Neutron diffraction data to a resolution of 2.1 Å has been collected using microgravity-grown perdeuterated TS crystals from PCG-15.
59 BASIC BIOLOGICAL SCIENCES↗
Structural Basis of the Stereochemistry of Inhibition of Tryptophan Synthase by Tryptophan and Deriv
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Phillips, Robert S.↗
Structural Snapshots of Proteus vulgaris Tryptophan Indole-Lyase Reveal Insights into the Catalytic Mechanism
Tryptophan indole lyase (TIL; [E.C. 4.1.99.1]) is a bacterial pyridoxal-5′-phosphate (PLP)-dependent enzyme that catalyzes reversible β-elimination of indole from L-tryptophan. The mechanism of elimination of indole from L-tryptophan starts with the formation of an external aldimine of the substrate and PLP, followed by deprotonation of the α-CH of the substrate, forming a resonance-stabilized quinonoid intermediate. Proton transfer to C3 of the indole ring and carbon–carbon bond cleavage of the quinonoid intermediate provide indole and aminoacrylate bound to PLP, which then releases indole, followed by iminopyruvate. We have now determined the X-ray crystal structures of TIL complexes with (3S)-dioxindolyl-l-alanine, an inhibitor, and with substrates L-tryptophan, 7-aza-L-tryptophan, and S-ethyl-l-cysteine (SEC) in the presence of benzimidazole (BZI), an isostere of the product indole. These structures show a mixture of gem-diamine, external aldimine, quinonoid, and aminoacrylate intermediates, in both open and closed active site conformations. In the closed conformations of L-tryptophan, (3S)-dioxindolyl-l-alanine, and 7-aza-L-tryptophan complexes, hydrogen bonds form between Asp-133 with N1 of the ligand heterocyclic ring and NE2 of His-458 in the small domain of TIL. This hydrogen bond also forms in the BZI complex with the aminoacrylate intermediates formed from both L-tryptophan and SEC. The closed quinonoid complex of 7-aza-L-tryptophan shows that the azaindole ring in the closed conformation is bent out of plane of the Cβ–C3 bond by about 40°, putting it in a geometry that leads toward the transition-state geometry. Thus, both conformational dynamics and substrate activation play critical roles in the reaction mechanism of the TIL.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Combining Random Mutagenesis and Metabolic Engineering for Enhanced Tryptophan Production in Synechocystis sp. Strain PCC 6803
Tryptophan (Trp) is an essential aromatic amino acid that has value as an animal feed supplement, as the amount found in plant-based sources is insufficient. An alternative to production by engineered microbial fermentation is to have tryptophan biosynthesized by a photosynthetic microorganism that could replace or supplement both the plant and industrially used microbes. We selected Synechocystis sp. strain PCC 6803, a model cyanobacterium, as the host and studied metabolic engineering and random mutagenesis approaches. Previous work on engineering heterotrophic microbes for improved Trp titers has targeted allosteric feedback regulation in enzymes 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase (DAHPS) and anthranilate synthase (AS) as major bottlenecks in the shikimate pathway. In this work, the genes encoding feedback-resistant enzymes from Escherichia coli, aroG fbr and trpE fbr , were overexpressed in the host wild-type (WT) strain. Separately, the WT strain was subjected to random mutagenesis and selection using an amino acid analog to isolate tryptophan-overproducing strains. The randomly mutagenized strains were sequenced in order to identify the mutations that resulted in the desirable phenotypes. Interestingly, the tryptophan overproducers had mutations in the gene encoding chorismate mutase (CM), which catalyzes the conversion of chorismate to prephenate. The best tryptophan overproducer from random mutagenesis was selected as a host for metabolic engineering where aroG fbr and trpE fbr were overexpressed. The best strain developed produced 212 ± 23 mg/liter of tryptophan after 10 days of photoautotrophic growth under 3% (vol/vol) CO2. We demonstrated that a combination of random mutagenesis and metabolic engineering was superior to either individual approach.
59 BASIC BIOLOGICAL SCIENCES↗
3D domain swapping in the TIM barrel of the α subunit of Streptococcus pneumoniae tryptophan synthase
Tryptophan synthase catalyzes the last two steps of tryptophan biosynthesis in plants, fungi and bacteria. It consists of two protein chains, designated α and β, encoded by trpA and trpB genes, that function as an αββα complex. Structural and functional features of tryptophan synthase have been extensively studied, explaining the roles of individual residues in the two active sites in catalysis and allosteric regulation. TrpA serves as a model for protein-folding studies. In 1969, Jackson and Yanofsky observed that the typically monomeric TrpA forms a small population of dimers. Dimerization was postulated to take place through an exchange of structural elements of the monomeric chains, a phenomenon later termed 3D domain swapping. The structural details of the TrpA dimer have remained unknown. Here, the crystal structure of the Streptococcus pneumoniae TrpA homodimer is reported, demonstrating 3D domain swapping in a TIM-barrel fold for the first time. The N-terminal domain comprising the H0–S1–H1–S2 elements is exchanged, while the hinge region corresponds to loop L2 linking strand S2 to helix H2′. The structural elements S2 and L2 carry the catalytic residues Glu52 and Asp63. As the S2 element is part of the swapped domain, the architecture of the catalytic apparatus in the dimer is recreated from two protein chains. The homodimer interface overlaps with the α–β interface of the tryptophan synthase αββα heterotetramer, suggesting that the 3D domain-swapped dimer cannot form a complex with the β subunit. In the crystal, the dimers assemble into a decamer comprising two pentameric rings.
3D domain swapping↗
The hydrogen bond between tryptophan and the host molecule induced fluorescence enhancement
Abstract Tryptophan is one of important amino acids in the human body, therefore its detection is particularly important. The 3,5‐bis(4‐pyridyl)‐4‐amino‐1,2,4‐triazole (BPAT) organic molecule was designed to be used as fluorescence detectors to detect tryptophan molecules for the interaction between the host and the guest. BPAT shows good sensitivity and selectivity towards tryptophan compared with other amino acid molecules. The limit of detection obtained from formula 3δ/K SV is considered to be 5.43 × 10 −7 mol/L. We speculated that this change is mainly caused by the hydrogen bond between tryptophan and the host molecule BPAT. This conjecture was verified by the controlled experiments with other host molecules.
Yang, Xi↗
Pyridine-containing substrate analogs are restricted from accessing the human cytochrome P450 8B1 active site by tryptophan 281
The human oxysterol 12α-hydroxylase cytochrome P450 8B1 (CYP8B1) is a validated drug target for both type 2 diabetes and nonalcoholic fatty liver disease, but effective selective inhibitors are not yet available. Herein, steroidal substrate-mimicking compounds with a pyridine ring appended to the C12 site of metabolism were designed as inhibitors, synthesized, and evaluated in terms of their functional and structural interactions with CYP8B1. While the pyridine nitrogen was intended to coordinate the CYP8B1 active site heme iron, none of these compounds elicited shifts in the CYP8B1 Soret absorbance consistent with this type of interaction. However, when CYP8B1 was cocrystallized with the pyridine-containing compound with the 3-keto-Δ4 steroid backbone most similar to the endogenous substrate, it was apparent that this ligand was bound in a channel leading to the active site, instead of near the heme iron. Inspection of this structure suggested that tryptophan 281 directly above the heme might restrict active site binding of potential inhibitors with this design. This hypothesis was supported when a CYP8B1 W281F mutation did allow all three compounds to coordinate the heme iron as designed. These results indicated that the design of next-generation CYP8B1 inhibitors should be compatible with the low-ceiling tryptophan immediately above the heme iron.
59 BASIC BIOLOGICAL SCIENCES↗
MYC transcription factors coordinate tryptophan–dependent defence responses and compromise seed yield in Arabidopsis
Robust plant immunity negatively affects other fitness traits, including growth and seed production. Jasmonate (JA) confers broad-spectrum protection against plant consumers by stimulating the degradation of JASMONATE ZIM-DOMAIN (JAZ) proteins, which in turn relieves repression on transcription factors (TFs) coincident with reduced growth and fecundity. The molecular mechanisms underlying JA-mediated decreases in fitness remain largely unknown. To assess the contribution of MYC TFs to growth and reproductive fitness at high levels of defence, we mutated three MYC genes in a JAZ-deficient mutant (jazD) of Arabidopsis thaliana that exhibits strong defence and low seed yield. Genetic epistasis analysis showed that de-repression of MYC TFs in jazD not only conferred strong resistance to insect herbivory but also reduced shoot and root growth, fruit size and seed yield. We also provided evidence that the JAZ–MYC module coordinates the supply of tryptophan with the production of indole glucosinolates and the proliferation of endoplasmic reticulum bodies that metabolise glucosinolates through the action of b-glucosidases. Our results establish MYCs as major regulators of growth- and reproductive–defence tradeoffs and further indicate that these factors coordinate tryptophan availability with the production of amino acid-derived defence compounds.
59 BASIC BIOLOGICAL SCIENCES↗
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 β-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 β-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid-base catalytic residue βLys87. 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 C $\bar{β}$ 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.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
An evolutionarily conserved tryptophan cage promotes folding of the extended RNA recognition motif in the hnRNPR ‐like protein family
Abstract The heterogeneous nuclear ribonucleoprotein (hnRNP) R‐like family is a class of RNA binding proteins in the hnRNP superfamily with diverse functions in RNA processing. Here, we present the 1.90 Å X‐ray crystal structure and solution NMR studies of the first RNA recognition motif (RRM) of human hnRNPR. We find that this domain adopts an extended RRM (eRRM1) featuring a canonical RRM with a structured N‐terminal extension (N ext ) motif that docks against the RRM and extends the β‐sheet surface. The adjoining loop is structured and forms a tryptophan cage motif to position the N ext motif for docking to the RRM. Combining mutagenesis, solution NMR spectroscopy, and thermal denaturation studies, we evaluate the importance of residues in the N ext –RRM interface and adjoining loop on eRRM folding and conformational dynamics. We find that these sites are essential for protein solubility, conformational ordering, and thermal stability. Consistent with their importance, mutations in the N ext –RRM interface and loop are associated with several cancers in a survey of somatic mutations in cancer studies. Sequence and structure comparison of the human hnRNPR eRRM1 to experimentally verified and predicted hnRNPR‐like proteins reveals conserved features in the eRRM.
Biochemistry & Molecular Biology↗
Neutron diffraction from a microgravity-grown crystal reveals the active site hydrogens of the internal aldimine form of tryptophan synthase
Pyridoxal 5′-phosphate (PLP), the biologically active form of vitamin B6, is an essential cofactor in many biosynthetic pathways. The emergence of PLP-dependent enzymes as drug targets and biocatalysts, such as tryptophan synthase (TS), has underlined the demand to understand PLP-dependent catalysis and reaction specificity. The ability of neutron diffraction to resolve the positions of hydrogen atoms makes it an ideal technique to understand how the electrostatic environment and selective protonation of PLP regulates PLP-dependent activities. Facilitated by microgravity crystallization of TS with the Toledo Crystallization Box, we report the 2.1 Å joint X-ray/neutron (XN) structure of TS with PLP in the internal aldimine form. Positions of hydrogens were directly determined in both the α- and β-active sites, including PLP cofactor. The joint XN structure thus provides insight into the selective protonation of the internal aldimine and the electrostatic environment of TS necessary to understand the overall catalytic mechanism.
59 BASIC BIOLOGICAL SCIENCES↗
Ultraviolet Superradiance from Mega-Networks of Tryptophan in Biological Architectures
Networks of tryptophan (Trp)–an aromatic amino acid with strong fluorescence response–are ubiquitous in biological systems, forming diverse architectures in transmembrane proteins, cytoskeletal filaments, subneuronal elements, photoreceptor complexes, virion capsids, and other cellular structures. We analyze the cooperative effects induced by ultraviolet (UV) excitation of several biologically relevant Trp mega-networks, thus giving insights into novel mechanisms for cellular signaling and control. Our theoretical analysis in the single-excitation manifold predicts the formation of strongly superradiant states due to collective interactions among organized arrangements of up to >10 5 Trp UV-excited transition dipoles in microtubule architectures, which leads to an enhancement of the fluorescence quantum yield (QY) that is confirmed by our experiments. We demonstrate the observed consequences of this superradiant behavior in the fluorescence QY for hierarchically organized tubulin structures, which increases in different geometric regimes at thermal equilibrium before saturation, highlighting the effect’s persistence in the presence of disorder. Our work thus showcases the many orders of magnitude across which the brightest (hundreds of femtoseconds) and darkest (tens of seconds) states can coexist in these Trp lattices.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Combining mechanistic and machine learning models for predictive engineering and optimization of tryptophan metabolism
Abstract Through advanced mechanistic modeling and the generation of large high-quality datasets, machine learning is becoming an integral part of understanding and engineering living systems. Here we show that mechanistic and machine learning models can be combined to enable accurate genotype-to-phenotype predictions. We use a genome-scale model to pinpoint engineering targets, efficient library construction of metabolic pathway designs, and high-throughput biosensor-enabled screening for training diverse machine learning algorithms. From a single data-generation cycle, this enables successful forward engineering of complex aromatic amino acid metabolism in yeast, with the best machine learning-guided design recommendations improving tryptophan titer and productivity by up to 74 and 43%, respectively, compared to the best designs used for algorithm training. Thus, this study highlights the power of combining mechanistic and machine learning models to effectively direct metabolic engineering efforts.
59 BASIC BIOLOGICAL SCIENCES↗
Sequence-based generative AI design of versatile tryptophan synthases
Enzymes are powerful and sustainable catalysts, but their widespread application is limited by the difficulty of identifying functional starting points for optimization, creating a major bottleneck in early- stage biocatalyst discovery. Designing libraries of such starting enzymes remains particularly challenging. Here, we use the GenSLM protein language model to generate novel β-subunit of tryptophan synthase (TrpB) enzymes that express in Escherichia coli and are both stable and catalytically active. Many generated TrpBs also display significant substrate promiscuity, outperforming their natural counterparts on non-native substrates. Some even surpass laboratory-evolved TrpBs. Comparison of the most-active and most-promiscuous generated TrpB to its closest natural homolog confirms that the enhanced versatility is absent from the natural enzyme, highlighting the creative potential of generative models. These results demonstrate that the generated TrpBs not only preserve natural structure and function but also acquire non-natural properties, establishing generative models as powerful tools for biocatalyst discovery and engineering.
biocatalysis↗
A single diiron enzyme catalyses the oxidative rearrangement of tryptophan to indole nitrile
Abstract Nitriles are uncommon in nature and are typically constructed from oximes through the oxidative decarboxylation of amino acid substrates or from the derivatization of carboxylic acids. Here we report a third nitrile biosynthesis strategy featuring the cyanobacterial nitrile synthase AetD. During the biosynthesis of the eagle-killing neurotoxin, aetokthonotoxin, AetD transforms the 2-aminopropionate portion of 5,7-dibromo-l-tryptophan to a nitrile. Employing a combination of structural, biochemical and biophysical techniques, we characterized AetD as a non-haem diiron enzyme that belongs to the emerging haem-oxygenase-like dimetal oxidase superfamily. High-resolution crystal structures of AetD together with the identification of catalytically relevant products provide mechanistic insights into how AetD affords this unique transformation, which we propose proceeds via an aziridine intermediate. Our work presents a unique template for nitrile biogenesis and portrays a substrate binding and metallocofactor assembly mechanism that may be shared among other haem-oxygenase-like dimetal oxidase enzymes.
Chemistry↗
Manipulating ultrafast even-order nonlinear chiral responses of L-tryptophan by polarization pulse shaping
Molecular chirality has long been monitored in the frequency domain in the ultraviolet, visible, and infrared regimes. Recently developed time-domain approaches can detect time-dependent chiral dynamics by enhancing intrinsically weak chiral signals. Even-order nonlinear signals in chiral molecules have gained attention thanks to their existence in the electric dipole approximation, without relying on the weaker higher-order multipole interactions. We illustrate the optimization of temporal polarization pulse-shaping in various frequency ranges (infrared/optical and optical/X ray) to enhance chiral nonlinear signals. These signals can be recast as an overlap integral of matter and field pseudoscalars which contain the relevant chiral information. Simulations are carried out for second- and fourth-order nonlinear spectroscopies in L-tryptophan.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Structure and Activity of the Thermophilic Tryptophan;#8208;6 Halogenase BorH
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Lingkon, Kazi↗