Tyrosine Phenol-lyase Research Articles

Serine 51 residue of Citrobacter freundii tyrosine phenol-lyase assists in C-α-proton abstraction and transfer in the reaction with substrate

In the spatial structure of tyrosine phenol-lyase, the Ser51 residue is located in the active site of the enzyme. The replacement of Ser51 with Ala by site-directed mutagenesis led to a decrease of the kcat/Km parameter for reactions with l-tyrosine and 3-fluoro-l-tyrosine by three orders of magnitude, compared to wild type enzyme. For the elimination reactions of S-alkylcysteines, the values of kcat/Km decreased by an average of two orders of magnitude. The results of spectral studies of the mutant enzyme gave evidence for a considerable change of the chiral properties of the active site as a result of the replacement. Fast kinetic studies for the complexes of the mutant form with competitive inhibitors allowed us to conclude that the Ser51 residue interacts with the side chain amino group of Lys257 at the stage of C-α-proton abstraction. This interaction ensures the correct orientation of the side chain of Lys257 accepting the C-α-proton of the external aldimine and stabilizes its ammonium form. Also, it is probable that Ser51 takes part in formation of a chain of hydrogen bonds which is necessary to perform the transfer of the C-α-proton to the C-4′-position of the leaving phenol group in the reaction with the natural substrate.

Biochemical characterization of a novel tyrosine phenol-lyase from Fusobacterium nucleatum for highly efficient biosynthesis of l-DOPA.

Tyrosine phenol-lyase (TPL) catalyzes the reversible cleavage of l-tyrosine to phenol, pyruvate and ammonia. When pyrocatechol is substituted for phenol, l-dihydroxyphenylalanine (l-DOPA) is produced. The TPL-catalyzed route was regarded as the most economic process for l-DOPA production. In this study, a novel TPL from Fusobacterium nucleatum (Fn-TPL) was successfully overexpressed in Escherichia coli and screened for l-DOPA synthesis with a specific activity of 2.69Umg-1. Fn-TPL was found to be a tetramer, and the optimal temperature and pH for α, β-elimination of l-tyrosine was 60°C and pH 8.5, respectively. The enzyme showed broad substrate specificity toward natural and synthetic l-amino acids. Kinetic analysis suggested that the kcat/Km value for l-tyrosine decomposition was much higher than that for l-DOPA decomposition, while Fn-TPL exhibited similar catalytic efficiency for synthesis of l-tyrosine and l-DOPA. With whole cells of recombinant E. coli as biocatalyst, l-DOPA yield reached 110gL-1 with a pyrocatechol conversion of 95%, which was comparable to the reported highest level. The results demonstrated the great potential of Fn-TPL for industrial production of l-DOPA.

Ground-State Destabilization by Phe-448 and Phe-449 Contributes to Tyrosine Phenol-Lyase Catalysis

The role of transition-state stabilization in enzyme catalysis, as proposed by Pauling, has been clearly demonstrated by extensive studies. In contrast, ground-state destabilization can also contribute to enzyme catalysis, but experimental evidence has been more limited. In recent years, high-resolution X-ray crystal structures of enzyme–substrate complexes have been obtained which show evidence for ground-state strain. We found that Y71F and F448H mutant tyrosine phenol-lyase (TPL) form complexes with 3-fluoro-l-tyrosine, a substrate, which shows a bending of the substrate aromatic ring about 20° out of plane, and we suggested that this was evidence for ground-state destabilization in TPL catalysis. Here, we have now evaluated quantitatively the role of ground-state destabilization in TPL catalysis. Phe-448 and Phe-449 are in close contact with the bound substrate side chain, and by mutating these residues to alanine and leucine, the contribution they play via ground-state destabilization was investigate...

Inhibition of tyrosine phenol-lyase by tyrosine homologues.

We have designed, synthesized, and evaluated tyrosine homologues and their O-methyl derivatives as potential inhibitors for tyrosine phenol lyase (TPL, E.C. 4.1.99.2). Recently, we reported that homologues of tryptophan are potent inhibitors of tryptophan indole-lyase (tryptophanase, TIL, E.C. 4.1.99.1), with K i values in the low µM range (Do et al. Arch Biochem Biophys 560:20-26, 2014). As the structure and mechanism for TPL is very similar to that of TIL, we postulated that tyrosine homologues could also be potent inhibitors of TPL. However, we have found that homotyrosine, bishomotyrosine, and their corresponding O-methyl derivatives are competitive inhibitors of TPL, which exhibit K i values in the range of 0.8-1.5mM. Thus, these compounds are not potent inhibitors, but instead bind with affinities similar to common amino acids, such as phenylalanine or methionine. Pre-steady-state kinetic data were very similar for all compounds tested and demonstrated the formation of an equilibrating mixture of aldimine and quinonoid intermediates upon binding. Interestingly, we also observed a blue-shift for the absorbance peak of external aldimine complexes of all tyrosine homologues, suggesting possible strain at the active site due to accommodating the elongated side chains.

Engineering and comparison of non-natural pathways for microbial phenol production.

The non-renewable petrochemical phenol is used as a precursor to produce numerous fine and commodity chemicals, including various pharmaceuticals and phenolic resins. Microbial phenol biosynthesis has previously been established, stemming from endogenous tyrosine via tyrosine phenol lyase (TPL). TPL, however, suffers from feedback inhibition and equilibrium limitations, both of which contribute to reduced flux through the overall pathway. To address these limitations, two novel and non-natural phenol biosynthesis pathways, both stemming instead from chorismate, were constructed and comparatively evaluated. The first proceeds to phenol in one heterologous step via the intermediate p-hydroxybenzoic acid, while the second involves two heterologous steps and the associated intermediates isochorismate and salicylate. Maximum phenol titers achieved via these two alternative pathways reached as high as 377 ± 14 and 259 ± 31 mg/L in batch shake flask cultures, respectively. In contrast, under analogous conditions, phenol production via the established TPL-dependent route reached 377 ± 23 mg/L, which approaches the maximum achievable output reported to date under batch conditions. Additional strain development and optimization of relevant culture conditions with respect to each individual pathway is ultimately expected to result in further improved phenol production. Biotechnol. Bioeng. 2016;113: 1745-1754. © 2016 Wiley Periodicals, Inc.

Two-step enzymatic synthesis of tyramine from raw pyruvate fermentation broth

Tyramine, as a metabolite of tyrosine, is an important intermediate in synthesizing some drugs and medicinal materials. In this study, an efficient method for producing tyramine was developed by a two-step biocatalytic reaction with recombinant tyrosine phenol-lyase whole cells and tyrosine decarboxylase immobilized cells. Raw pyruvate fermentation broth was used as substrate of tyrosine phenol-lyase to economically produce l-tyrosine. l-tyrosine was catalyzed by immobilized tyrosine decarboxylase cells to effectively synthesize tyramine. The conditions of two-step enzymatic catalysis reactions were optimized separately, and the influence of immobilization on tyrosine decarboxylase activity was investigated. In a scale up study, 94.3% l-tyrosine was obtained from raw pyruvate fermentation broth under the optimal conditions. l-Tyrosine was decarboxylated to tyramine with a high yield 91.2%. The total yield of tyramine could reach approximately 86% by this two-step biocatalytic reaction. This study provides us with a green strategy for efficient preparation of tyramine.

Biocatalytic One-Pot Synthesis of l-Tyrosine Derivatives from Monosubstituted Benzenes, Pyruvate, and Ammonia

l-Tyrosine derivatives were obtained in >97% ee via a biocatalytic one-pot two-step cascade using substituted benzenes, pyruvate, and NH3 as starting materials. In the first step, monosubstituted arenes were regioselectively hydroxylated in the o-position by monooxygenase P450 BM3 (using O2 as oxidant with NADPH-recycling) to yield the corresponding phenols, which subsequently underwent C–C coupling and simultaneous asymmetric amination with pyruvate and NH3 using tyrosine phenol lyase to furnish l-DOPA surrogates in up to 5.2 g L–1. Instead of analytically pure arenes, crude aromatic gasoline blends containing toluene were used to yield 3-methyl-l-tyrosine in excellent yield (2 g L–1) and >97% ee.

Three-step biocatalytic reaction using whole cells for efficient production of tyramine from keratin acid hydrolysis wastewater.

Tyramine has been paid more attention in recent years as a significant metabolite of tyrosine and catecholamine drug and an intermediate of medicinal material and some drugs. In this study, an effective, green, and three-step biocatalytic synthesis method for production of tyramine starting from serine in keratin acid hydrolysis wastewater was developed and investigated. Serine deaminase from Escherichia coli was first combined with tyrosine phenol-lyase from Citrobacter koseri, to convert L-serine to L-tyrosine. L-Tyrosine can then be decarboxylated to tyramine by tyrosinede carboxylase from Lactobacillus brevis. All these enzymes originated from recombinant whole cells. Serine deaminaseand tyrosine phenol-lyase could efficiently convert L-serine in wastewater to L-tyrosine at pH 8.0, 37 °C, and Triton X-100 of 0.04% when tyrosine phenol-lyase and its corresponding substrates were sequentially added. Tyrosine conversion rate reached 98 % by L-tyrosine decarboxylase. In scale-up study, the conversion yield of L-serine in wastewater to tyrosine was up to 89 %. L-Tyrosine was decarboxylated to tyramine with a high yield 94 %. Tyramine hydrochloride was obtained with a total yield 84 %. This study has provided an efficient way of recycling keratin acid hydrolysis wastewater to produce tyramine.

An Optimized Protocol for Bacterial Cell Disruption with Zirconium Beads and Recovery of Intracellular Tyrosine Phenol Lyase

The biotechnological production of intracellular tyrosine phenol lyase finds important applications in pharmaceutical industry for the development of novel therapeutic molecules like L-DOPA. Cell disruption is one of the important steps in the downstream processing of intracellular enzymes and bioprocess industry demands this operation to be energy, cost and time efficient. The efficiency of the disruption process for the release of tyrosine phenol lyase from Citrobacter freundii in a bead beater with zirconium beads was studied. Under optimum conditions of cell disruption (0.1mm zirconium beads, 6mg/mL dcw cells, 1mM EDTA, 0.7mM DTT and 9 disruption cycle), the specific activity of enzyme reached to 0.038 IU/mg. The stability of cell free tyrosine phenol lyase increased by 5.8-fold by addition of EDTA and DTT. The disruption of C. freundii cells with beads seems to be effective as evidenced by transmission electron microscopy. This cell free enzyme with enhanced stability can be used subsequently in suspension or immobilized form for the biotransformtion reactions. Keywords: Cell disruption, intracellular enzymes, tyrosine phenol lyase, Citrobacter freundii, bead beater, TEM.

Construction of a novel phenol synthetic pathway in Escherichia coli through 4-hydroxybenzoate decarboxylation.

Phenol is a bulk chemical with lots of applications in the chemical industry. Fermentative production of phenol had been realized in both Pseudomonas putida and Escherichia coli by recruiting tyrosine phenol-lyase (TPL). The TPL pathway needs tyrosine as the direct precursor for phenol production. In this work, a novel phenol synthetic pathway was created in E. coli by recruiting 4-hydroxybenzoate decarboxylase, which can convert 4-hydroxybenzoate to phenol and carbon dioxide. Activating 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase and chorismate pyruvate lyase (UbiC) through plasmid overexpression led to 7- and 69-fold increase of phenol production, respectively, demonstrating that these two enzymes were the rate-limiting steps for phenol production. Genetically stable strains were then obtained by gene integration and gene modulation directly in chromosome. Phenol titer increased 147-fold (from 1.7 to 250 mg/L) after modulating the DAHP synthase, UbiC, and 4-hydroxybenzoate decarboxylase genes in chromosome. Five solvents were tested for two-phase extractive fermentation to eliminate phenol toxicity to E. coli cells. Tributyrin and dibutyl phthalate were the best two solvents for improving phenol production, leading to 23 and 30 % increase of total phenol production, respectively. Two-phase fed-batch fermentation of the best strain Phe009 was performed in a 7 L fermentor, which produced 9.51 g/L phenol with a yield of 0.061 g/g glucose.

Structures of Escherichia coli tryptophanase in holo and 'semi-holo' forms.

Two crystal forms of Escherichia coli tryptophanase (tryptophan indole-lyase, Trpase) were obtained under the same crystallization conditions. Both forms belonged to the same space group P43212 but had slightly different unit-cell parameters. The holo crystal form, with pyridoxal phosphate (PLP) bound to Lys270 of both polypeptide chains in the asymmetric unit, diffracted to 2.9 Å resolution. The second crystal form diffracted to 3.2 Å resolution. Of the two subunits in the asymmetric unit, one was found in the holo form, while the other appeared to be in the apo form in a wide-open conformation with two sulfate ions bound in the vicinity of the active site. The conformation of all holo subunits is the same in both crystal forms. The structures suggest that Trpase is flexible in the apo form. Its conformation partially closes upon binding of PLP. The closed conformation might correspond to the enzyme in its active state with both cofactor and substrate bound in a similar way as in tyrosine phenol-lyase.

Overview on the biotechnological production of L-DOPA.

L-DOPA (3,4-dihydroxyphenyl-L-alanine) has been widely used as a drug for Parkinson's disease caused by deficiency of the neurotransmitter dopamine. Since Monsanto developed the commercial process for L-DOPA synthesis for the first time, most of currently supplied L-DOPA has been produced by the asymmetric method, especially asymmetric hydrogenation. However, the asymmetric synthesis shows critical limitations such as a poor conversion rate and a low enantioselectivity. Accordingly, alternative biotechnological approaches have been researched for overcoming the shortcomings: microbial fermentation using microorganisms with tyrosinase, tyrosine phenol-lyase, or p-hydroxyphenylacetate 3-hydroxylase activity and enzymatic conversion by immobilized tyrosinase. Actually, Ajinomoto Co. Ltd commercialized Erwinia herbicola fermentation to produce L-DOPA from catechol. In addition, the electroenzymatic conversion system was recently introduced as a newly emerging scheme. In this review, we aim to not only overview the biotechnological L-DOPA production methods, but also to briefly compare and analyze their advantages and drawbacks. Furthermore, we suggest the future potential of biotechnological L-DOPA production as an industrial process.

The role of substrate strain in the mechanism of the carbon–carbon lyases

The carbon–carbon lyases, tryptophan indole lyase (TIL) and tyrosine phenol-lyase (TPL) are bacterial enzymes which catalyze the reversible elimination of indole and phenol from l-tryptophan and l-tyrosine, respectively. These PLP-dependent enzymes show high sequence homology (∼40% identity) and both form homotetrameric structures. Steady state kinetic studies with both enzymes show that an active site base is essential for activity, and α-deuterated substrates exhibit modest primary isotope effects on kcat and kcat/Km, suggesting that substrate deprotonation is partially rate-limiting. Pre-steady state kinetics with TPL and TIL show rapid formation of external aldimine intermediates, followed by deprotonation to give quinonoid intermediates absorbing at about 500nm. In the presence of phenol and indole analogues, 4-hydroxypyridine and benzimidazole, the quinonoid intermediates of TPL and TIL decay to aminoacrylate intermediates, with λmax at about 340nm. Surprisingly, there are significant kinetic isotope effects on both formation and subsequent decay of the quinonoid intermediates when α-deuterated substrates are used. The crystal structure of TPL with a bound competitive inhibitor, 4-hydroxyphenylpropionate, identified several essential catalytic residues: Tyr-71, Thr-124, Arg-381, and Phe-448. The active sites of TIL and TPL are highly conserved with the exceptions of these residues: Arg-381(TPL)/Ile-396 (TIL); Thr-124 (TPL)/Asp-137 (TIL), and Phe-448 (TPL)/His-463 (TIL). Mutagenesis of these residues results in dramatic decreases in catalytic activity without changing substrate specificity. The conserved tyrosine, Tyr-71 (TPL)/Tyr-74 (TIL) is essential for elimination activity with both enzymes, and likely plays a role as a proton donor to the leaving group. Mutation of Arg-381 and Thr-124 of TPL to alanine results in very low but measurable catalytic activity. Crystallography of Y71F and F448H TPL with 3-fluoro-l-tyrosine bound demonstrated that there are two quinonoid structures, relaxed and tense. In the relaxed structure, the substrate aromatic ring is in plane with the Cβ–Cγ bond, but in the tense structure, the substrate aromatic ring is about 20° out of plane with the Cβ–Cγ bond. In the tense structure, hydrogen bonds are formed between the substrate OH and the guanidinium of Arg-381 and the OH of Thr-124, and the phenyl rings of Phe-448 and 449 provide steric strain. Based on the effects of mutagenesis, the substrate strain is estimated to contribute about 108 to TPL catalysis. Thus, the mechanisms of TPL and TIL require both substrate strain and acid/base catalysis, and substrate strain is probably responsible for the very high substrate specificity of TPL and TIL.

Toward a Generalized and High-throughput Enzyme Screening System Based on Artificial Genetic Circuits

Large-scale screening of enzyme libraries is essential for the development of cost-effective biological processes, which will be indispensable for the production of sustainable biobased chemicals. Here, we introduce a genetic circuit termed the Genetic Enzyme Screening System that is highly useful for high-throughput enzyme screening from diverse microbial metagenomes. The circuit consists of two AND logics. The first AND logic, the two inputs of which are the target enzyme and its substrate, is responsible for the accumulation of a phenol compound in cell. Then, the phenol compound and its inducible transcription factor, whose activation turns on the expression of a reporter gene, interact in the other logic gate. We confirmed that an individual cell harboring this genetic circuit can present approximately a 100-fold higher cellular fluorescence than the negative control and can be easily quantified by flow cytometry depending on the amounts of phenolic derivatives. The high sensitivity of the genetic circuit enables the rapid discovery of novel enzymes from metagenomic libraries, even for genes that show marginal activities in a host system. The crucial feature of this approach is that this single system can be used to screen a variety of enzymes that produce a phenol compound from respective synthetic phenyl-substrates, including cellulase, lipase, alkaline phosphatase, tyrosine phenol-lyase, and methyl parathion hydrolase. Consequently, the highly sensitive and quantitative nature of this genetic circuit along with flow cytometry techniques could provide a widely applicable toolkit for discovering and engineering novel enzymes at a single cell level.

Metabolic engineering ofEscherichia colifor the production of phenol from glucose

Phenol is an industrially versatile commodity chemical and is currently produced from fossil resources. Phenol's biological production from renewable resources has been limited due to its toxicity to microorganisms. Here, we simultaneously engineered 18 Escherichia coli strains for the production of phenol using synthetic regulatory small RNA (sRNA) technology. sRNA-based knock-down of the two regulators and overexpression of the genes involved in the tyrosine biosynthetic pathway together with tyrosine phenol-lyase (TPL) in E. coli strains resulted in the production of phenol from glucose. The 18 engineered E. coli strains showed significant differences in the production of tyrosine (i.e. the immediate precursor for phenol), TPL activity, and tolerance to phenol. Among the engineered E. coli strains, the BL21 strain produced phenol most efficiently: 419 mg/L by flask culture and 1.69 g/L by fed-batch culture. The final titer and productivity were further improved through biphasic fed-batch fermentation using glycerol tributyrate as an extractant of phenol. The concentration of phenol in the glycerol tributyrate phase and fermentation broth reached 9.84 and 0.3 g/L, respectively, in 21 hours, which translates into the final phenol titer and productivity of 3.79 g/L and 0.18 g/L/h, respectively. This is the highest titer achieved by microbial fermentation. Although further engineering is required to be competitive with the current petro-based process, the strategies used for the development of the engineered strain and fermentation process will provide a valuable framework for the microbial production of toxic chemicals.

Purification and Characterization of Tyrosine Phenol Lyase from Citrobacter freundii

The purification and characterization of intracellular tyrosine phenol lyase from Citrobacter freundii has been carried out. The enzyme was purified 35-fold to homogeneity by ammonium sulphate precipitation and hydrophobic interaction chromatography. Its subunit molecular weight was found to be 52 kDa on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The purified tyrosine phenol lyase showed maximum activity in borate buffer (0.05 M at pH 8.5) at 45 °C after 20 min of incubation. The Km and Vmax values of purified enzyme were found to be 0.446 mm and 0.342 mM/min/mg. This enzyme exhibits t1/2 of 10, 52 and 130 min at 55, 45 and 35 °C, respectively. The N-terminal amino acid sequence was determined as MET-ASN-TYR-PRO-ALA-GLU-PRO-PHE-ARG-ILETRP- TRP-VAL-GLY.

Paraffin as oxygen vector modulates tyrosine phenol lyase production byCitrobacter freundiiMTCC 2424

The efficiency of three oxygen-vectors liquid paraffin, silicone oil and n-dodecane in the production of tyrosine phenol lyase (TPL) by Citrobacter freundii MTCC 2424 was evaluated at 4% (v/v) concentration. The liquid paraffin as oxygenvectors was found to exhibit a stimulatory effect on TPL synthesis. The liquid paraffin at 6% (v/v) resulted in 34% increase in the TPL synthesis accompanied by a 13% increase in the production of cell mass at a 10 L scale. This improvement in TPL and cell mass production in the presence of liquid paraffin can be related to the fact that liquid paraffin was capable of maintaining dissolved O2 concentration above 28% throughout the course of the fermentation. Maintenance of the dissolved O2 concentration above 28% could be viewed in terms of an adequate oxygen supply to the rapidly dividing cells of the bacterium, which in turn resulted in enhanced synthesis of TPL and cell mass.

Crystal Structure of Citrobacter freundii Asp214Ala Tyrosine Phenollyase Reveals that Asp214 is Critical for Maintaining a Strain in the Internal Aldimine

Tyrosine phenol-lyase (TPL) is a pyridoxal-5′-phosphate (PLP) dependent enzyme which catalyzes β-elimination of L-tyrosine. In the holoenzyme the protonated pyridinium N1 atom of the PLP cofactor is hydrogen-bonded to the side chain of Asp214. Here we report the X-ray structure of C. freundii D214A TPL determined at 1.9 A resolution. Comparison with the structure of the wild-type TPL shows that the D214A replacement induced significant conformational reorganization in the active site leading to its partial closure. Significantly, in D214A TPL the strain in the internal aldimine is completely released and the pyridine N1 atom of PLP is deprotonated. These observations explain the considerably reduced activity of the D214A TPL towards its substrates [T. V. Demidkina et al., Biochim. Biophys. Acta, Proteins Proteomics 1764 (2006) 1268–1276]. The reported structure reveals that Asp214 is critical for maintaining the strain in the internal aldimine. We argue that this strain is used by the enzyme to accelerate the transaldimination reaction, the first step in the enzymatic catalysis.

Crystal Structure of E. coli Tryptophanase in “Semi-Holo” Form: An Insight into Allostery of the Enzyme

A new crystal form of E. coli tryptophanase (tryptophan indole-lyase, Trpase) (space group P43212, a=b=109.97 A, c=238.40 A) was obtained under the same conditions as the tetragonal crystals of holo E. coli. Trpase. The structure was solved by molecular replacement at 3.2 A resolution using the coordinates of apo E. coli Trpase (PDB code 2OQX) as a search model and refined to R=21.3 %, Rfree=28.9 %. Out of two polypeptide chains contained in the asymmetric unit, one was found in holo form with PLP covalently attached to Lys271, while the other appeared to be in the apo form. The overall conformation of the holo subunit is the same as in the holo form of tyrosine phenol-lyase. The apo subunit is found in a wide-open conformation very similar to the one observed in the crystal structure of apo Trpase. Taking into account the flexibility of apo Trpase as seen in the known structures and difference in the crystallization conditions (pH, precipitant) and crystal packing, this finding is quite unexpected. We suggest that apo Trpase is found in the solution predominantly in the wide-open conformation which partially closes upon binding of PLP. The closed conformation might correspond to the enzyme state with both cofactor and substrate bound, in a way similar to tyrosine phenol-lyase. In addition, the conformation of the loop 301-310 is different in apo and holo subunits of the new structure suggesting that this conformational change is not induced by the oxidation of Cys298.

Crystallographic snapshots of tyrosine phenol-lyase show that substrate strain plays a role in C-C bond cleavage.

The key step in the enzymatic reaction catalyzed by tyrosine phenol-lyase (TPL) is reversible cleavage of the Cβ–Cγ bond of l-tyrosine. Here, we present X-ray structures for two enzymatic states that form just before and after the cleavage of the carbon–carbon bond. As for most other pyridoxal 5′-phosphate-dependent enzymes, the first state, a quinonoid intermediate, is central for the catalysis. We captured this relatively unstable intermediate in the crystalline state by introducing substitutions Y71F or F448H in Citrobacter freundii TPL and briefly soaking crystals of the mutant enzymes with a substrate 3-fluoro-l-tyrosine followed by flash-cooling. The X-ray structures, determined at ∼2.0 Å resolution, reveal two quinonoid geometries: “relaxed” in the open and “tense” in the closed state of the active site. The “tense” state is characterized by changes in enzyme contacts made with the substrate’s phenolic moiety, which result in significantly strained conformation at Cβ and Cγ positions. We also captured, at 2.25 Å resolution, the X-ray structure for the state just after the substrate’s Cβ–Cγ bond cleavage by preparing the ternary complex between TPL, alanine quinonoid and pyridine N-oxide, which mimics the α-aminoacrylate intermediate with bound phenol. In this state, the enzyme–ligand contacts remain almost exactly the same as in the “tense” quinonoid, indicating that the strain induced by the closure of the active site facilitates elimination of phenol. Taken together, structural observations demonstrate that the enzyme serves not only to stabilize the transition state but also to destabilize the ground state.