Characterization of aminoacrylate intermediates of pyridoxal-5'-phosphate dependent enzymes.

Pyridoxal-5'-phosphate (PLP) Schiff's bases of 2-aminoacrylate are intermediates in ß-elimination and ß-substitution reaction of PLP-dependent enzymes. These enzymes are found in two major families, the α-, or aminotransferase, superfamily, and the ß-family. While the α-family enzymes primarily catalyze ß-eliminations, the ß-family enzymes catalyze both ß-elimination and ß-substitution reactions. Tyrosine phenol-lyase (TPL), which catalyzes the reversible elimination of phenol from l-tyrosine, is an example of an α-family enzyme. Tryptophan synthase catalyzes the irreversible formation of l-tryptophan from l-serine and indole, and is an example of a ß-family enzyme. The identification and characterization of aminoacrylate intermediates in the reactions of both of these enzymes is discussed. The use of UV-visible absorption and fluorescence spectroscopy, X-ray and neutron crystallography, and NMR spectroscopy to identify aminoacrylate intermediates in these and other PLP enzymes is presented.

M379A Mutant Tyrosine Phenol-lyase from Citrobacter freundii Has Altered Conformational Dynamics.

The M379A mutant of Citrobacter freundii tyrosine phenol-lyase (TPL) has been prepared. M379A TPL is a robust catalyst to prepare a number of tyrosines substituted at the 3-position with bulky groups that cannot be made with wild type TPL. The three dimensional structures of M379A TPL complexed with L-methionine and 3-bromo-DL-phenylalanine have been determined by X-ray crystallography. Methionine is bound as a quinonoid complex in a closed active site in 3 of 4 chains of homotetrameric M379A TPL. M379A TPL reacts with L-methionine about 8-fold slower than wild type TPL. The temperature dependence shows that the slower reaction is due to less positive activation entropy. The structure of the M379A TPL complex of 3-bromo-DL-phenylalanine has a quinonoid complex in two subunits, with an open active site conformation. The effects of the M379A mutation on TPL suggest that the mutant enzyme has altered the conformational dynamics of the active site.

Regulating the biosynthesis of pyridoxal 5'-phosphate with riboswitch to enhance L-DOPA production by Escherichia coli whole-cell biotransformation.

Pyridoxal 5'-phosphate (PLP) is an essential cofactor that participates in ∼4% enzymatic activities cataloged by the Enzyme Commission. The intracellular level of PLP is usually lower than that demanded in industrial catalysis. To realize the self-supply of PLP cofactor in whole-cell biotransformation, the de novo ribose 5-phosphate (R5P)-dependent PLP synthesis pathway was constructed. The pdxST genes from Bacillus subtilis 168 were introduced into the tyrosine phenol-lyase (TPL)-overexpressing Escherichia coli BL21(DE3) strain. TPL and PdxST were co-expressed with a double-promoter or a compatible double-plasmid system. The 3,4-dihydroxyphenylacetate-L-alanine (L-DOPA) titer did not increase with the increase in the intracellular PLP concentration in these strains with TPL and PdxST co-expression. Therefore, it is necessary to optimize the intracellular PLP metabolism level so as to achieve a higher L-DOPA titer and avoid the formation of L-DOPA-PLP cyclic adducts. The thi riboswitch binds to PLP and forms a complex such that the ribosome cannot have access to the Shine-Dalgarno (SD) sequence. Therefore, this metabolite-sensing regulation system was applied to regulate the translation of pdxST mRNA. Riboswitch was introduced into pET-TPL-pdxST-2 to downregulate the expression of PdxST and biosynthesis of PLP at the translation level by sequestering the ribosome-binding site. As a result, the titer and productivity of L-DOPA using the strain BL21-TPLST-Ribo1 improved to 69.8 g/L and 13.96 g/L/h, respectively, with a catechol conversion of 95.9% and intracellular PLP accumulation of 24.8 µM.

Purification and Biochemical Characterization of a Tyrosine Phenol-lyase from Morganella morganii.

Tyrosine phenol-lyase (TPL) is a valuable and cost-effective biocatalyst for the biosynthesis of L-tyrosine and its derivatives, which are valuable intermediates in the pharmaceutical industry. A TPL from Morganella morganii (Mm-TPL) was overexpressed in Escherichia coli and characterized. Mm-TPL was determined as a homotetramer with molecular weight of 52 kDa per subunit. Its optimal temperature and pH for ß-elimination of L-tyrosine were 45 °C and pH 8.5, respectively. Mm-TPL manifested strict substrate specificity for the reverse reaction of ß-elimination and ortho- and meta-substituted phenols with small steric size were preferred substrates. The enzyme showed excellent catalytic performance for synthesis of L-tyrosine, 3-fluoro-L-tyrosine, and L-DOPA with a yield of 98.1%, 95.1%, and 87.2%, respectively. Furthermore, the fed-batch bioprocess displayed space-time yields of 9.6 g L-1 h-1 for L-tyrosine and 4.2 g L-1 h-1 for 3-fluoro-L-tyrosine with a yield of 67.4 g L-1 and 29.5 g L-1, respectively. These results demonstrated the great potential of Mm-TPL for industrial application.

Site-directed mutagenesis to improve the thermostability of tyrosine phenol-lyase.

3,4-Dihydroxyphenyl-L-alanine (L-DOPA) is the most important antiparkinsonian drug, and tyrosine phenol-lyase (TPL)-based enzyme catalysis process is one of the most adopted methods on industrial scale production. TPL activity and stability represent the rate-limiting step in L-DOPA synthesis. Here, 25 TPL mutants were predicted, and two were confirmed as exhibiting the highest L-DOPA production and named E313W and E313M. The L-DOPA production from E313W and E313M was 47.5 g/L and 62.1 g/L, which was 110.2 % and 174.8 % higher, respectively, than that observed from wild-type (WT) TPL. The Km of E313W and E313M showed no apparent decrease, whereas the kcat of E313W and E313M improved by 45.5 % and 36.4 %, respectively, relative to WT TPL. Additionally, E313W and E313M displayed improved thermostability, a higher melting temperature, and enhanced affinity between for pyridoxal-5'-phosphate. Structural analysis of the mutants suggested increased stability of the N-terminal region via enhanced interactions between the mutated residues and H317. Application of these mutants in a substrate fed-batch strategy as whole-cell biocatalysts allows realization of a cost-efficient short fermentation period resulting in high L-DOPA yield.

An efficient colorimetric high-throughput screening method for synthetic activity of tyrosine phenol-lyase.

Tyrosine phenol-lyase (TPL) naturally catalyzes the reversible ß-elimination of l-tyrosine to phenol, pyruvate and ammonium. With its reverse reaction (synthetic activity), l-tyrosine and its derivatives could be synthesized with high atom economy, which are widely used in pharmaceutical industries. In this study, a high-throughput screening method for synthetic activity of TPL was developed. One of the substrate, sodium pyruvate was found to react with salicylaldehyde under alkali condition, forming a yellow color compound. The concentration of sodium pyruvate can be quantified according to the absorbance of the colorimetric compound at wavelength of 465 nm and the activity of TPL could be screened according to the decrease of the absorbance. After optimization of the colorimetric reaction conditions, the established high-throughput screening method was successfully used for screening of TPL with enhanced activity for l-DOPA synthesis. The confirmed sensitivity and accuracy demonstrated the feasibility and application potential of this screening method.

Crystal Structures of Wild-Type and F448A Mutant Citrobacter freundii Tyrosine Phenol-Lyase Complexed with a Substrate and Inhibitors: Implications for the Reaction Mechanism.

Tyrosine phenol-lyase (TPL; EC 4.1.99.2) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the reversible hydrolytic cleavage of l-tyrosine to phenol and ammonium pyruvate. We have shown previously that F448A TPL has kcat and kcat/ Km values for l-tyrosine reduced by ∼104-fold [Phillips, R. S., Vita, A., Spivey, J. B., Rudloff, A. P., Driscoll, M. D., and Hay, S. (2016) ACS Catal. 6, 6770-6779]. We have now obtained crystal structures of F448A TPL and complexes with l-alanine, l-methionine, l-phenylalanine, and 3-F-l-tyrosine at 2.05-2.27 Å and the complex of wild-type TPL with l-phenylalanine at 1.8 Å. The small domain of F448A TPL, where Phe-448 is located, is more disordered in chain A than in wild-type TPL. The complexes of F448A TPL with l-alanine and l-phenylalanine are in an open conformation in both chains, while the complex with l-methionine is a 52:48 open:closed equilibrium mixture in chain A. Wild-type TPL with l-alanine is closed in chain A and open in chain B, and the complex with l-phenylalanine is a 56:44 open:closed mixture in chain A. Thus, the Phe-448 to alanine mutation affects the conformational equilibrium of open and closed active sites. The structure of the 3-F-l-tyrosine quinonoid complex of F448A TPL is unstrained and in an open conformation, with a hydrogen bond from the phenolic OH to Thr-124. These results support our previous conclusion that ground-state strain plays a critical role in the mechanism of TPL.

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, ß-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.

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.

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 0.8-1.5 mM. 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.

Chemistry and diversity of pyridoxal-5'-phosphate dependent enzymes.

Pyridoxal-5'-phosphate (PLP) is a versatile cofactor that enzymes use to catalyze a wide variety of reactions of amino acids, including transamination, decarboxylation, racemization, ß- and γ-eliminations and substitutions, retro-aldol and Claisen reactions. These reactions depend on the ability of PLP to stabilize, to a varying degree, α-carbanionic intermediates. Furthermore, oxidative decarboxylations and rearrangements suggest that PLP can stabilize radical intermediates as well. The reaction mechanisms of two PLP-dependent enzymes are discussed, kynureninase and tyrosine phenol-lyase (TPL). Kynureninase catalyzes a retro-Claisen reaction of kynurenine to give anthranilate and alanine. The key step, hydration of the γ-carbonyl, is assisted by acid-base catalysis with the phosphate of the PLP, mediated by a conserved tyrosine, and an oxyanion hole. TPL catalyzes the reversible elimination of phenol, a poor leaving group, from l-tyrosine. In TPL, the Cß-Cγ bond cleavage is accelerated by ground state strain from the bending of the substrate ring out of the plane with the Cß-Cγ bond. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

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 Cß-Cγ bond, but in the tense structure, the substrate aromatic ring is about 20° out of plane with the Cß-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 10(8) 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.

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.

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 Cß-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 Cß and Cγ positions. We also captured, at 2.25 Å resolution, the X-ray structure for the state just after the substrate's Cß-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.

Development of anaerobically inducible nar promoter expression vectors for the expression of recombinant proteins in Escherichia coli.

Dissolved oxygen (DO)-controlled nar promoter expression vectors were constructed, and their expression efficiency was compared with that of the T7 promoter pET22 expression vector by expressing human growth hormone (hGH), enhanced green fluorescence protein (EGFP), and ß-tyrosinase in Escherichia coli cells. The nar promoter expression vector pRBS, which was engineered with a 5'-untranslated region and ribosomal binding site for the T7 promoter, expressed hGH at a rate of up to 32% of the total cellular proteins (TCP) in E. coli W3110narL⁻. The expression level of hGH was further enhanced, up to ~42% of the TCP, by adding the N-terminal peptide tag of ß-galactosidase to hGH, which was comparable to the expression of ~43% of the TCP in pET-lac:hGH/BL21(DE3). A further engineered expression vector, pRBS(fnr), which coexpressed fumarate/nitrate reductase (fnr), expressed more EGFP than pET22 in BL21(DE3). In addition, recombinant ß-tyrosinase was successfully expressed at a rate of up to ~45% of the TCP in pRBS(fnr) in W3110narL⁻. From these results, the DO-controlled nar promoter system developed in this study can be considered a reliable and cost-effective expression system for protein production, especially in large-scale fermentation, as an alternative to the pET/BL(DE3) system.

Stereospecificity of isotopic exchange of C-α-protons of glycine catalyzed by three PLP-dependent lyases: the unusual case of tyrosine phenol-lyase.

A comparative study of the kinetics and stereospecificity of isotopic exchange of the pro-2R- and pro-2S protons of glycine in (2)H(2)O under the action of tyrosine phenol-lyase (TPL), tryptophan indole-lyase (TIL) and methionine γ-lyase (MGL) was undertaken. The kinetics of exchange was monitored using both (1)H- and (13)C-NMR. In the three compared lyases the stereospecificities of the main reactions with natural substrates dictate orthogonal orientation of the pro-2R proton of glycine with respect to the cofactor pyridoxal 5'-phosphate (PLP) plane. Consequently, according to Dunathan's postulate with all the three enzymes pro-2R proton should exchange faster than does the pro-2S one. In fact the found ratios of 2R:2S reactivities are 1:20 for TPL, 108:1 for TIL, and 1,440:1 for MGL. Thus, TPL displays an unprecedented inversion of stereospecificity. A probable mechanism of the observed phenomenon is suggested, which is based on the X-ray data for the quinonoid intermediate, formed in the reaction of TPL with L-alanine. The mechanism implies different conformational changes in the active site upon binding of glycine and alanine. These changes can lead to relative stabilization of either the neutral amino group, accepting the α-proton, or the respective ammonium group, which is formed after the proton abstraction.

Simultaneous improvement of catalytic activity and thermal stability of tyrosine phenol-lyase by directed evolution.

The tyrosine phenol-lyase from Symbiobacterium toebii was engineered to improve both its stability and catalytic activity by the application of random mutagenesis and subsequent reassembly of the acquired mutations. Activity screening of the random library produced four mutants with a two-fold improved activity, whereas parallel screening after heat treatment at 65 degrees C identified three mutants with half-inactivation temperatures improved by up to 5.6 degrees C. The selected mutants were then reassembled using the staggered extension PCR method, and subsequent screening of the library produced seven mutants with up to three-fold improved activity and half-inactivation temperatures improved by up to 11.2 degrees C. Sequence analyses revealed that the stability-improved hits included A13V, E83K and T407A mutations, whereas the activity-improved hits included the additional T129I or T451A mutation. In particular, the A13V mutation was propagated in the hits with improved stability during the reassembly-screening process, indicating the critical nature of the N-terminal moiety for enzyme stability. Furthermore, homology modeling of the enzyme structure revealed that most of the stability mutations were located around the dimer-dimer interface, including the N-terminus, whereas the activity-improving mutations were located further away, thereby minimizing any interference that would be detrimental to the co-improvement of the stability and catalytic activity of the enzyme.

[Spatial structure and mechanism of tyrosine phenol-lyase and tryptophan indole-lyase].

The bacterial tyrosine phenol-lyase (EC 4.1.99.2) and tryptoptophan indole-lyase (EC 4.1.99.1) belong to pyridoxal-5'-phosphate dependent beta-eliminating lyases, catalysing the reversible decomposition of L-tyrosine and L-tryptophan to pyruvate, ammonia, and phenol or indole correspondingly. Data on the three dimentional structures of the holoenzymes of tyrosine phenol-lyase and tryptophan indole-lyase and several enzyme-inhibitor complexes, modeling distinct reaction stages of the beta-elimination of L-tyrosine are described in the paper and structural bases of monovalent cations influence of activity of the enzymes are discussed. The spectral and catalytic properties of the mutant enzymes were studied. The data thus obtained have allowed us to elucidate the catalytic functions of a number of amino acid residues and conclude that the acid-base properties of the catalytic groups of the enzymes under the optimal for the catalysis conditions in hydrophobic active sites of tyrosine phenol-lyase and tryptoptophan indol-lyase are different from those in water solutions. Study of the mechanisms of labilization of Calpha-proton of the bound amino acids and activation of the leaving groups of the substrates during the catalytic process has demonstrated that in certain cases concerted reaction pathways are realized instead of stepwise ones.

Insights into the catalytic mechanism of tyrosine phenol-lyase from X-ray structures of quinonoid intermediates.

Amino acid transformations catalyzed by a number of pyridoxal 5'-phosphate (PLP)-dependent enzymes involve abstraction of the Calpha proton from an external aldimine formed between a substrate and the cofactor leading to the formation of a quinonoid intermediate. Despite the key role played by the quinonoid intermediates in the catalysis by PLP-dependent enzymes, limited accurate information is available about their structures. We trapped the quinonoid intermediates of Citrobacter freundii tyrosine phenol-lyase with L-alanine and L-methionine in the crystalline state and determined their structures at 1.9- and 1.95-A resolution, respectively, by cryo-crystallography. The data reveal a network of protein-PLP-substrate interactions that stabilize the planar geometry of the quinonoid intermediate. In both structures the protein subunits are found in two conformations, open and closed, uncovering the mechanism by which binding of the substrate and restructuring of the active site during its closure protect the quinonoid intermediate from the solvent and bring catalytically important residues into positions suitable for the abstraction of phenol during the beta-elimination of L-tyrosine. In addition, the structural data indicate a mechanism for alanine racemization involving two bases, Lys-257 and a water molecule. These two bases are connected by a hydrogen bonding system allowing internal transfer of the Calpha proton.