Search

Enzymes with high catalytic activity and stability are essential for industrial production, yet most natural enzymes do not meet these requirements. Therefore, efficient strategies for enzyme engineering are crucial. In this study, we developed a cost-effective computational design strategy to enhance the activity of tyrosine phenol-lyase (TPL) for the production of L-DOPA. By integrating structural analysis with computational design, and guided by our understanding of conformational flexibility of TPL, we identified a region where enhanced stability is most likely to facilitate enzyme activity. We screened stabilizing mutations by Cartesian_ddg in Rosetta. After identifying single stabilizing mutations, we grouped the nearby positions harboring multiple stabilizing mutations and calculated the energy of combinatorial variants. We found two promising groups where most variants exhibited lower calculated energy than the wild-type. Experimental validation showed five variants in these groups exhibit increased activity, with the two best variants showing catalytic activity enhancements of 1.8-fold and 1.6-fold compared to the wild-type enzyme., (© 2024. The Author(s).)

Pyridoxal phosphate (PLP), the active form of vitamin B6, is an important coenzyme in various enzyme-catalyzed reactions. PLP-dependent enzymes can catalyze a variety of chemical reactions, such as racemization, decarboxylation, β-addition, β-elimination, retro-aldol cleavage, transamination, and α-elimination. They are biologically synthesized a powerful tool for a variety of natural amino acids, non-natural amino acids and their related compounds. This article details the structural features and catalytic mechanisms of typical PLP-dependent enzymes such as ω-transaminase, lysine decarboxylase, threonine aldolase, and L-tyrosine phenol-lyase, and reviews the research progress in molecular modification and industrial applications of these enzymes. Finally, this article provides an outlook on the future development of PLP-dependent enzymes, including in vivo regeneration system and industrial applications of PLP cofactors, and discusses the tremendous potential of these enzymes in biocatalytic applications.

Tyrosine phenol lyase (TPL) synthesises L-tyrosine derivatives from monophenols, pyruvate and ammonia. Production of such high-value aromatic chemicals from biomass-derived raw materials is of great interest. In this study, six monophenols (guaiacol, phenol, o-cresol, m-cresol, catechol and syringol) were chosen based on the structure of lignin and were studied as substrates in the enzymatic reaction. Single monophenol reactions (SMR) and binary monophenol reactions (BMR) with guaiacol were carried out. TPL-M379V was found to be selective towards guaiacol (84.5 % conv.). The highest single activity was measured towards phenol (93.9 % conv.). However, the enzyme preferred guaiacol over phenol in the BMRs. Syringol was found to be inert in the reaction, whereas catechol had an inhibitory effect on the enzymatic reaction, in addition to causing degradation of all the substrates in the medium. Doubling the guaiacol concentration in the SMR did not significantly increase the production of 3-O-methyldopa (conv. 45.9 %). However, in the binary reaction systems the total monophenol conversions were higher with guaiacol and phenol (total 62.4 %) or o-cresol (total 57.1 %). This indicates possible substrate/product specific inhibition. The study provides new data on activity, selectivity and inhibitory effects of monophenols in the synthetic reaction catalysed by TPL-M379V, especially in mixed-substrate reactions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Aromatic amino acids and their derivatives are diverse primary and secondary metabolites with critical roles in protein synthesis, cell structure and integrity, defense and signaling. All de novo aromatic amino acid production relies on a set of ancient and highly conserved chemistries. Here we introduce a new enzymatic transformation for L-tyrosine synthesis by demonstrating that the β-subunit of tryptophan synthase-which natively couples indole and L-serine to form L-tryptophan-can act as a latent 'tyrosine synthase'. A single substitution of a near-universally conserved catalytic residue unlocks activity toward simple phenol analogs and yields exclusive para carbon-carbon bond formation to furnish L-tyrosines. Structural and mechanistic studies show how a new active-site water molecule orients phenols for a nonnative mechanism of alkylation, with additional directed evolution resulting in a net >30,000-fold rate enhancement. This new biocatalyst can be used to efficiently prepare valuable L-tyrosine analogs at gram scales and provides the missing chemistry for a conceptually different pathway to L-tyrosine., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Pyridoxal-5'-phosphate (PLP) Schiff's bases of 2-aminoacrylate are intermediates in β-elimination and β-substitution reaction of PLP-dependent enzymes. These enzymes are found in two major families, the α-, or aminotransferase, superfamily, and the β-family. While the α-family enzymes primarily catalyze β-eliminations, the β-family enzymes catalyze both β-elimination and β-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 β-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., (Copyright © 2023 Elsevier Inc. All rights reserved.)

L-DOPA is an amino acid that is used as a treatment for Parkinson's disease. A simple enzymatic synthesis method of L-DOPA had been developed using bacterial L-tyrosine phenol-lyase (Tpl). This review describes research on screening of bacterial strains, culture conditions, properties of the enzyme, reaction mechanism of the enzyme, and the reaction conditions for the production of L-DOPA. Furthermore, molecular bleeding of constitutively Tpl-overproducing strains is described, which were developed based on mutations in a DNA binding protein, TyrR, which controls the induction of tpl gene expression.

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., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Tyrosine phenol-lyase (TPL) exhibits great potential in industrial biosynthesis of l-tyrosine and its derivates. To uncover and screen TPLs with excellent catalytic properties, there is unmet demand for development of facile and reliable screening system for TPL. Here we presented a novel assay format for the detection of TPL activity based on catechol 2,3-dioxygenase (C23O)-catalyzed reaction. Catechol released from TPL-catalyzed cleavage of 3,4-dihydroxy-l-phenylalanine (l-DOPA) was further oxidized by C23O to form 2-hydroxymuconate semialdehyde, which could be readily detected by spectrophotometric measurements at 375 nm. The assay achieved a unique balance between the ease of operation and superiority of analytical performances including linearity, sensitivity and accuracy. In addition, this assay enabled real-time monitoring of TPL activity with high efficiency and reliability. As C23O is highly specific towards catechol, a non-natural product of microorganism, the assay was therefore accessible to both crude cell extracts and the whole-cell system without elaborate purification steps of enzymes, which could greatly expedite discovery and engineering of TPLs. This study provided fundamental principle for high-throughput screening of other enzymes consuming or producing catechol derivatives., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Objective: To solve the bottleneck of plasmid instability during microbial fermentation of L-DOPA with recombinant Escherichia coli expressing heterologous tyrosine phenol lyase., Results: The tyrosine phenol lyase from Fusobacterium nucleatum was constitutively expressed in E. coli and a fed-batch fermentation process with temperature down-shift cultivation was performed. Efficient strategies including replacing the original ampicillin resistance gene, as well as inserting cer site that is active for resolving plasmid multimers were applied. As a result, the plasmid stability was increased. The co-use of cer site on plasmid and kanamycin in culture medium resulted in proportion of plasmid containing cells maintained at 100% after fermentation for 35 h. The specific activity of tyrosine phenol lyase reached 1493 U/g dcw, while the volumetric activity increased from 2943 to 14,408 U/L for L-DOPA biosynthesis., Conclusions: The established strategies for plasmid stability is not only promoted the applicability of the recombinant cells for L-DOPA production, but also provides important guidance for industrial fermentation with improved microbial productivity.

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., (Copyright © 2020. Published by Elsevier B.V.)

The formation of inclusion bodies (IBs) without enzyme activity in bacterial research is generally undesirable. Researchers have attempted to recovery the enzyme activities of IBs, which are commonly known as active IBs. Tyrosine phenol-lyase (TPL) is an important enzyme that can convert pyruvate and phenol into 3,4-dihydroxyphenyl-L-alanine (L-DOPA) and IBs of TPL can commonly occur. To induce the correct folding and recover the enzyme activity of the IBs, peptides, such as ELK16, DKL6, L6KD, ELP10, ELP20, L6K2, EAK16, 18A, and GFIL16, were fused to the carboxyl terminus of TPL. The results showed that aggregate particles of TPL-DKL6, TPL-ELP10, TPL-EAK16, TPL-18A, and TPL-GFIL16 improved the enzyme activity by 40.9%, 50.7%, 48.9%, 86.6%, and 97.9%, respectively. The peptides TPL-DKL6, TPL-EAK16, TPL-18A, and TPL-GFIL16 displayed significantly improved thermostability compared with TPL. L-DOPA titer of TPL-ELP10, TPL-EAK16, TPL-18A, and TPL-GFIL16, with cells reaching 37.8 g/L, 53.8 g/L, 37.5 g/L, and 29.1 g/L, had an improvement of 111%, 201%, 109%, and 63%, respectively. A higher activity and L-DOPA titer of the TPL-EAK16 could be valuable for its industrial application to biosynthesize L-DOPA.

Genetic circuit-based biosensors have emerged as an effective analytical tool in synthetic biology; these biosensors can be applied to high-throughput screening of new biocatalysts and metabolic pathways. Sigma 54 (σ 54 )-dependent transcription factor (TF) can be a valuable component of these biosensors owing to its intrinsic silent property compared to most of the housekeeping sigma 70 (σ 70 ) TFs. Here, we show that these unique characteristics of σ 54 -dependent TFs can be used to control the host cell state to be more appropriate for high-throughput screening. The acclimation of cell state was achieved by using guanosine (penta)tetraphosphate ((p)ppGpp)-related genes (relA, spoT) and nutrient conditions, to link the σ 54 TF-based reporter expression with the target enzyme activity. By controlling stringent programmed responses and optimizing assay conditions, catalytically improved tyrosine phenol lyase (TPL) enzymes were successfully obtained using a σ 54 -dependent DmpR as the TF component, demonstrating the practical feasibility of this biosensor. This combinatorial strategy of biosensors using σ factor-dependent TFs will allow for more effective high-throughput enzyme engineering with broad applicability.

Herein we report the development of an efficient cellular system for the in vivo biosynthesis of Tyr-analogs and their concurrent incorporation into target proteins by the residue-specific approach. This system makes use of common phenol derivatives and the tyrosine phenol lyase machinery to create various tyrosine analogues that impart desired properties on the target proteins. Biosynthesized 2-fluorotyrosine was incorporated into three industrially important enzymes which resulted in enhanced thermostability.

L-tyrosine is an amino acid that has been widely used in the food, agriculture and pharmaceutical industries. In order to screen a tyrosine phenol-lyase (TPL) with excellent catalytic performance for L-tyrosine production, TPL genes from Citrobacter freundii (CfTPL), Erwinia herbicola (EhTPL) and Rhodobacter capsulatus (TutA) were codon-optimized and overexpressed in Escherichia coli. The results showed that EhTPL had the highest whole cell catalysis activity and tyrosine yield (3-fold that of CfTPL). The results of RT-qPCR and a stability analysis also revealed that EhTPL had a higher transcriptional level in whole cell catalysis, while CfTPL possessed greater stability. Conditions for the production by whole cell transformation were optimized in terms of reaction conditions and fed-batch strategy. Finally, the maximum production was obtained with a titer of 48.5 g·L -1 by intermittent feeding with a conversion ratio of 75%., (Copyright © 2019. Published by Elsevier Inc.)

L-DOPA is a key pharmaceutical agent for treating Parkinson's, and market demand has exploded due to the aging population. There are several challenges associated with the chemical synthesis of L-DOPA, including complicated operation, harsh conditions, and serious pollution. A biocatalysis route for L-DOPA production is promising, especially via a route catalyzed by tyrosine phenol lyase (TPL). In this study, using TPL derived from Erwinia herbicola (Eh-TPL), a mutant Eh-TPL was obtained by integrating enzyme evolution and high-throughput screening methods. L-DOPA production using recombinant Escherichia coli BL21 (DE3) cells harbouring mutant Eh-TPL was enhanced by 36.5% in shake flasks, and the temperature range and alkali resistance of the Eh-TPL mutant were promoted. Sequence analysis revealed two mutated amino acids in the mutant (S20C and N161S), which reduced the length of a hydrogen bond and generated new hydrogen bonds. Using a fed-batch mode for whole-cell catalysis in a 5 L bioreactor, the titre of L-DOPA reached 69.1 g L -1 with high productivity of 11.52 g L -1  h -1 , demonstrating the great potential of Eh-TPL variants for industrial production of L-DOPA.

D-danshensu (D-DSS), extracted from the plant Salvia miltiorrhiza (Danshen), is widely used to treat cardiovascular and cerebrovascular diseases. Here we engineered Escherichia coli strains to produce D-DSS from catechol, pyruvate and ammonia by one-pot biotransformation. Tyrosin-phenol lyase (TPL), L-amino acid deaminase (aadL), D-lactate dehydrogenase (ldhD) and glucose dehydrogenase (gdh) genes were overexpressed in Escherichia coli strain. First, the expression of genes was regulated by different copy number plasmids combination, the result of E. coli TALG6, with strong overexpression of TPL, aadL, ldhD and moderate overexpression of gdh, exhibited 253.7% increase D-DSS production compared to E. coli TALG1. Second, the optimum concentration of catechol was found to be 50 mM. Finally, a fed-batch biotransformation strategy was proposed, namely the amount of catechol was added to 50 mM every 2 h. The total production of D-DSS reached 55.35 mM within 14 h, which was 1.7 times that without feeding., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Diabetic kidney disease is a major cause of renal failure that urgently necessitates a breakthrough in disease management. Here we show using untargeted metabolomics that levels of phenyl sulfate, a gut microbiota-derived metabolite, increase with the progression of diabetes in rats overexpressing human uremic toxin transporter SLCO4C1 in the kidney, and are decreased in rats with limited proteinuria. In experimental models of diabetes, phenyl sulfate administration induces albuminuria and podocyte damage. In a diabetic patient cohort, phenyl sulfate levels significantly correlate with basal and predicted 2-year progression of albuminuria in patients with microalbuminuria. Inhibition of tyrosine phenol-lyase, a bacterial enzyme responsible for the synthesis of phenol from dietary tyrosine before it is metabolized into phenyl sulfate in the liver, reduces albuminuria in diabetic mice. Together, our results suggest that phenyl sulfate contributes to albuminuria and could be used as a disease marker and future therapeutic target in diabetic kidney disease.

The radical S-adenosylmethionine (SAM) superfamily is currently the largest known enzyme family. These enzymes reductively cleave SAM to produce a highly reactive 5'-deoxyadenosyl (dAdo) radical, which abstracts a hydrogen from the substrate and initiates diverse reactions. The canonic dAdo radical-mediated hydrogen abstraction can be changed to radical addition reactions by using olefin-containing substrate analogues, which result in adenosylation reactions. Here we report investigation of the adenosylation reactions catalyzed by four radical SAM l-Tyr lyases (RSTLs), including HydG, FbiC, and two ThiH enzymes from different organisms. We show RSTLs have diverse substrate specificity, and ThiH from E. coli exhibits the highest substrate tolerance toward the tested substrates. We also show ThiH from Clostridium berjerinckii does not act on 4-amino-l-phenylalanine, but catalyzes adenosylation of the corresponding olefin-containing analogue, suggesting adenosylation may occur more easily than the canonic radical SAM reactions. Our study highlights the remarkable catalytic promiscuity of radical SAM enzyme and the potential in using these enzymes for the synthesis of nucleotide-containing compounds.

Tyrosine phenol lyase (TPL) is a robust biocatalyst for the production of L-dihydroxyphenylalanine (L-DOPA). The improvement of TPL production is conducive to the industrial potential. In this study, the optimization of culture medium of recombinant Escherichia coli harboring TPL from Fusobacterium nucleatum (Fn-TPL) was carried out. Sucrose and combination of yeast extract and peptone were selected as carbon and nitrogen source, respectively. Their optimal concentrations were determined by Box-Behnken design and the synergistic effect between yeast extract and peptone was found to be significant, with p-value < 0.05. The DO-STAT fed-batch fermentation under optimized culture condition was established and the oxygen level was fixed at 20%. Both the biomass and Fn-TPL activity were significantly increased, which were 35.6 g dcw/L and 12292 U/L, respectively. The results obtained significantly promote the industrial production of L-DOPA production.

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 k cat and k cat / K m values for l-tyrosine reduced by ∼10 4 -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.

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 k cat /K m 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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Aromatic chemicals are important building blocks for the production of a multitude of everyday commodities. Currently, aromatics production relies almost exclusively on petrochemical processes. To achieve sustainability, alternative synthesis methods need to be developed. Here, we strived for an efficient production of phenol, a model aromatic compound of industrial relevance, from renewable carbon sources using the solvent-tolerant biocatalyst Pseudomonas taiwanensis VLB120. First, multiple catabolic routes for the degradation of aromatics and related compounds were inactivated, thereby obtaining the chassis strain P. taiwanensis VLB120Δ5 incapable of growing on 4-hydroxybenzoate (ΔpobA), tyrosine (Δhpd), and quinate (ΔquiC, ΔquiC1, ΔquiC2). In this context, a novel gene contributing to the quinate catabolism was identified (quiC2). Second, we employed a combination of reverse- and forward engineering to increase metabolic flux towards the product, using leads obtained from the analysis of aromatics producing Pseudomonas putida strains previously generated by mutagenesis. Phenol production was enabled by the heterologous expression of a codon-optimized and chromosomally integrated tyrosine phenol-lyase encoding gene from Pantoea agglomerans AJ2985 (PaTPL2). The genomic modification of endogenous genes encoding TrpE P290S , AroF-1 P148L , and PheA T310I , and the deletion of pykA improved phenol production 17-fold, while also minimizing the burden caused by plasmids and auxotrophies. The additional overexpression of known bottleneck enzymes (AroG fbr , TyrA fbr ) derived from Escherichia coli further enhanced phenol titers. The best producing strain P. taiwanensis VLB120Δ5-TPL36 reached yields of 15.8% and 18.5% (Cmol/Cmol) phenol from glucose and glycerol, respectively, in a mineral medium without addition of complex nutrients. This is the highest yield ever reported for microbially produced phenol., (Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

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 k cat /K m 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 k cat /K m 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., (Copyright © 2017. Published by Elsevier B.V.)

Genetic circuit-based biosensors are useful in detecting target metabolites or in vivo enzymes using transcription factors (Tx) as a molecular switch to express reporter signals, such as cellular fluorescence and antibiotic resistance. Herein, a phenol-detecting Tx (DmpR) was employed as a critical tool for enzyme engineering, specifically for the rapid analysis of numerous mutants with multiple mutations at the active site of tryptophan-indole lyase (TIL, EC 4.1.99.1). Cellular fluorescence was monitored cell-by-cell using flow cytometry to detect the creation of phenolic compounds by a new tyrosine-phenol-lyase (TPL, EC 4.1.99.2). In the TIL scaffold, target amino acids near the indole ring (Asp 137 , Phe 304 , Val 394 , Ile 396 and His 463 ) were mutated randomly to construct a large diversity of specificity variations. Collection of candidate positives by cell sorting using flow cytometry and subsequent shuffling of beneficial mutations identified a critical hit with four mutations (D137P, F304D, V394L, and I396R) in the TIL sequence. The variant displayed one-thirteenth the level of TPL activity, compared with native TPLs, and completely lost the original TIL activity. The findings demonstrate that hypersensitive, Tx-based biosensors could be useful critically to generate new activity from a related template, which would alleviate the current burden to high-throughput screening.

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., (© 2016 Wiley Periodicals, Inc.)

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 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 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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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.

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.

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.

The δ(18)O value of the p-hydroxy group of L-tyrosine depends on the biosynthesis by plants or animals, respectively. In animal proteins it reflects the diet and is therefore an absolute indicator for illegal feeding with meat and bone meal. The aim of this investigation was to perform the positional (18)O determination on L-tyrosine via a one-step enzymatic degradation. Proteins from plants, herbivores, omnivores, and carnivores were characterized by their δ(13)C, δ(15)N, and δ(18)O values, the latter for normalizing the positional δ(18)O values. Their L-tyrosine was degraded by tyrosine phenol lyase to phenol, analyzed as (2,4,6)-tribromophenol. Degradation by tyrosine decarboxylase yielded tyramine. The δ(18)O values of both analytes corresponded to the trophic levels of their sources but were not identical, probably due to an isotope effect on the tyrosine phenol lyase reaction. Availability of the enzyme, easy control of the reaction, and isolation of the analyte are in favor of tyrosine decarboxylase degradation as a routine method.

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 β-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 β-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 β-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., (© 2010 Elsevier B.V. All rights reserved.)

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.

Given the well-established beneficial effects of Vitreoscilla hemoglobin (VHb) on heterologous organisms, the potential of this protein for the production of L-DOPA and dopamine in two bacteria, Citrobacter freundii and Erwinia herbicola, was investigated. The constructed recombinants bearing the VHb gene (vgb(+)) had substantially higher levels of cytoplasmic L-DOPA (112 mg/L for C. freundii and 97 mg/L for E. herbicola) than their respective hosts (30.4 and 33.8 mg/L) and the vgb(-) control strains (35.6 and 35.8 mg/L). Further, the vgb(+) recombinants of C. freundii and E. herbicola had 20-fold and about two orders of magnitude higher dopamine levels than their hosts, repectively. The activity of tyrosine phenol-lyase, the enzyme converting L-tyrosine to L-DOPA, was well-correlated to cytoplasmic L-DOPA levels. As cultures aged, higher tyrosine phenol-lyase activity of the vgb(+) strains was more apparent.

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.

The unknown genetic basis for improved phenol production by a recombinant Pseudomonas putida S12 derivative bearing the tpl (tyrosine-phenol lyase) gene was investigated via comparative transcriptomics, nucleotide sequence analysis, and targeted gene disruption. We show upregulation of tyrosine biosynthetic genes and possibly decreased biosynthesis of tryptophan caused by a mutation in the trpE gene as the genetic basis for the enhanced phenol production. In addition, several genes in degradation routes connected to the tyrosine biosynthetic pathway were upregulated. This either may be a side effect that negatively affects phenol production or may point to intracellular accumulation of tyrosine or its intermediates. A number of genes identified by the transcriptome analysis were selected for targeted disruption in P. putida S12TPL3. Physiological and biochemical examination of P. putida S12TPL3 and these mutants led to the conclusion that the metabolic flux toward tyrosine in P. putida S12TPL3 was improved to such an extent that the heterologous tyrosine-phenol lyase enzyme had become the rate-limiting step in phenol biosynthesis.

The aromatic amino acid L-tyrosine is used as a dietary supplement and has promise as a valuable precursor compound for various industrial and pharmaceutical applications. In contrast to chemical production, biotechnological methods can produce L-tyrosine from biomass feedstocks under environmentally friendly and near carbon-free conditions. In this minireview, various strategies for synthesizing L-tyrosine by employing biocatalysts are discussed, including initial approaches as well as more recent advances. Whereas early attempts to engineer L-tyrosine-excreting microbes were based on auxotrophic and antimetabolite-resistant mutants, recombinant deoxyribonucleic acid technology and a vastly increasing knowledge of bacterial physiology allowed recently for more targeted genetic manipulations and strain improvements. As an alternative route, L-tyrosine can also be obtained from the conversion of phenol, pyruvate, and ammonia or phenol and serine in reactions catalyzed by the enzyme tyrosine phenol lyase.

An efficient enzyme system for the synthesis of L-tyrosine was developed using a fed-batch reactor with continuous feeding of phenol, pyruvate, and ammonia. A thermo- and chemostable tyrosine phenol-lyase from Symbiobacterium toebii was employed as the biocatalyst in this work. The enzyme was produced using a constitutive expression system in Escherichia coli BL21, and prepared as a soluble extract by rapid clarification, involving treatment with 40% methanol in the presence of excess ammonium chloride. The stability of the enzyme was maintained for at least 18 h under the synthesis conditions, including 75 mM phenol at pH 8.5 and 40 degrees C. The fed-batch system (working volume, 0.5 1) containing 1.0 kU of the enzyme preparation was continuously fed with two substrate preparations: one containing 2.2 M phenol and 2.4 M sodium pyruvate, and the other containing 0.4 mM pyridoxal-5-phosphate and 4 M ammonium chloride (pH 8.5). The system produced 130 g/l of L-tyrosine within 30 h, mostly as precipitated particles, upon continuous feeding of the substrates for 22 h. The maximum conversion yield of L-tyrosine was 94% on the basis of the supplied phenol.

Citrobacter freundiil-tyrosine phenol-lyase (TPL) was inactivated by a Pictet-Spengler reaction between the cofactor and a substrate, 3,4-dihydroxyphenyl-L-alanine (L-dopa), in proportion to an increase in the reaction temperature. Random mutagenesis of the tpl gene resulted in the generation of a Thr15 to Ala mutant (T15A), which exhibited a two-fold improved activity towards L-DOPA as the substrate. The Thr15 residue was located on the intertwined N-terminal arm of the TPL structure, and comprised an H-bond network in proximity to the hydrophobic core between the catalytic dimers. The maximum activity of the mutant and native enzymes with L-DOPA was detected at 45 and 40 degrees C, respectively, which was 15 degrees C lower than when using L-tyrosine as the substrate. The half-lives at 45 degrees C were about 16.8 and 6.4 min for the mutant and native enzymes, respectively, in 10 mM L-DOPA. On treatment with excess pyridoxal-5'-phosphate (PLP), the L-DOPA-inactivated enzymes recovered over 80% of their original activities, thereby attributing the inactivation to a loss of the cofactor through Pictet-Spengler condensation with L-DOPA. Consistent with the extended half-life, the apparent Michaelis constant of the T15A enzyme for PLP (K(m,PLP)) increased slowly when increasing the temperature, while that of the native enzyme showed a sharp increase at temperatures higher than 50 degrees C, implying that the loss of the cofactor with the Pictet-Spengler reaction was prevented by the tighter binding and smaller release of the cofactor in the mutant enzyme.

Tyrosine phenol-lyase (TPL) from Citrobacter freundii is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the reversible hydrolytic cleavage of l-Tyr to give phenol and ammonium pyruvate. The proposed reaction mechanism for TPL involves formation of an external aldimine of the substrate, followed by deprotonation of the alpha-carbon to give a quinonoid intermediate. Elimination of phenol then has been proposed to give an alpha-aminoacrylate Schiff base, which releases iminopyruvate that ultimately undergoes hydrolysis to yield ammonium pyruvate. Previous stopped-flow kinetic experiments have provided direct spectroscopic evidence for the formation of the external aldimine and quinonoid intermediates in the reactions of substrates and inhibitors; however, the predicted alpha-aminoacrylate intermediate has not been previously observed. We have found that 4-hydroxypyridine, a non-nucleophilic analogue of phenol, selectively binds and stabilizes aminoacrylate intermediates in reactions of TPL with S-alkyl-l-cysteines, l-tyrosine, and 3-fluoro-l-tyrosine. In the presence of 4-hydroxypyridine, a new absorption band at 338 nm, assigned to the alpha-aminoacrylate, is observed with these substrates. Formation of the 338 nm peaks is concomitant with the decay of the quinonoid intermediates, with good isosbestic points at approximately 365 nm. The value of the rate constant for aminoacrylate formation is similar to k(cat), suggesting that leaving group elimination is at least partially rate limiting in TPL reactions. In the reaction of S-ethyl-l-cysteine in the presence of 4-hydroxypyridine, a subsequent slow reaction of the alpha-aminoacrylate is observed, which may be due to iminopyruvate formation. Both l-tyrosine and 3-fluoro-l-tyrosine exhibit kinetic isotope effects of approximately 2-3 on alpha-aminoacrylate formation when the alpha-(2)H-labeled substrates are used, consistent with the previously reported internal return of the alpha-proton to the phenol product. These results are the first direct spectroscopic observation of alpha-aminoacrylate intermediates in the reactions of TPL.

In the X-ray structure of tyrosine phenol-lyase (TPL) Asp214 is located at H-bonding distance from the N1 atom of the cofactor. This residue has been replaced with Ala and Asn and the properties of the mutant enzymes have been studied. The substitutions result in a decrease in the cofactor affinity of about four orders of magnitude. D214A and D214N TPLs do not catalyze the decomposition of l-Tyr and 3-fluoro-l-Tyr. They decompose substrates, containing better leaving groups with rates reduced by one or two orders of magnitude. Lognormal resolution of the spectra of the mutant enzymes revealed that the N1 atom of the cofactor is deprotonated. Spectral characteristics of internal and external aldimines of the mutant TPLs and the data on their interaction with quasisubstrates demonstrate that replacements of Asp214 lead to alteration of active site conformations. The mutant enzymes do not form noticeable amounts of a quinonoid upon interaction with inhibitors, but catalyze isotope exchange of C-alpha-proton of a number of amino acids for deuterium in (2)H(2)O. The k(ex) values for the isotope exchange of l-phenylalanine and 3-fluoro-l-tyrosine are close to the k(cat) values for reacting substrates. Thus, for the mutant TPLs the stage of C-alpha-proton abstraction may be considered as a rate-limiting for the whole reaction.

Tyrosine phenol-lyase, a tetrameric pyridoxal 5'-phosphate dependent enzyme, catalyzes the reversible hydrolytic cleavage of L-tyrosine to phenol and ammonium pyruvate. Here we describe the crystal structure of the Citrobacter freundii holoenzyme at 1.9 A resolution. The structure reveals a network of protein interactions with the cofactor, pyridoxal 5'-phosphate, and details of coordination of the catalytically important K+ ion. We also present the structure of the apoenzyme at 1.85 A resolution. Both structures were determined using crystals grown at pH 8.0, which is close to the pH of the maximal enzymatic activity (8.2). Comparison of the apoenzyme structure with the one previously determined at pH 6.0 reveals significant differences. The data suggest that the decrease of the enzymatic activity at pH 6.0 may be caused by conformational changes in the active site residues Tyr71, Tyr291, and Arg381 and in the monovalent cation binding residue Glu69. Moreover, at pH 8.0 we observe two different active site conformations: open, which was characterized before, and closed, which is observed for the first time in beta-eliminating lyases. In the closed conformation a significant part of the small domain undergoes an extraordinary motion of up to 12 A toward the large domain, closing the active site cleft and bringing the catalytically important Arg381 and Phe448 into the active site. The closed conformation allows rationalization of the results of previous mutational studies and suggests that the observed active site closure is critical for the course of the enzymatic reaction and for the enzyme's specificity toward its physiological substrate. Finally, the closed conformation allows us to model keto(imino)quinonoid, the key transition intermediate.

Efficient bioconversion of glucose to phenol via the central metabolite tyrosine was achieved in the solvent-tolerant strain Pseudomonas putida S12. The tpl gene from Pantoea agglomerans, encoding tyrosine phenol lyase, was introduced into P. putida S12 to enable phenol production. Tyrosine availability was a bottleneck for efficient production. The production host was optimized by overexpressing the aroF-1 gene, which codes for the first enzyme in the tyrosine biosynthetic pathway, and by random mutagenesis procedures involving selection with the toxic antimetabolites m-fluoro-dl-phenylalanine and m-fluoro-l-tyrosine. High-throughput screening of analogue-resistant mutants obtained in this way yielded a P. putida S12 derivative capable of producing 1.5 mM phenol in a shake flask culture with a yield of 6.7% (mol/mol). In a fed-batch process, the productivity was limited by accumulation of 5 mM phenol in the medium. This toxicity was overcome by use of octanol as an extractant for phenol in a biphasic medium-octanol system. This approach resulted in accumulation of 58 mM phenol in the octanol phase, and there was a twofold increase in the overall production compared to a single-phase fed batch.

The enzymatic production of 3,4-dihydroxyphenyl-L-alanine (L-DOPA) using Erwinia herbicola cells involves the action of tyrosine phenol-lyase (Tpl, EC 4.1.99.2). Since Tpl is only synthesized under L-tyrosine-induced conditions, the addition of L-tyrosine to the medium is unavoidable when preparing cells (the enzyme source), but severely impedes the pure preparation of the final product L-DOPA. We circumvented this problem by using recombinant E. herbicola cells carrying a mutant transcriptional regulator TyrR, which is capable of activating the tpl promoter in the absence of L-tyrosine.

Tyrosine phenol-lyase (TPL) from Citrobacter freundii is dependent on monovalent cations, K(+) or NH(4)(+), for high activity. We have shown previously that Glu-69, which is a ligand to the bound cation, is important in monovalent cation binding and activation [Sundararaju, B., Chen, H., Shillcutt, S., and Phillips, R. S. (2000) Biochemistry 39, 8546-8555]. Lys-256 is located in the monovalent cation binding site of TPL, where it forms a hydrogen bond with a structural water bound to the cation. This lysine residue is highly conserved in sequences of TPL and the paralogue, tryptophan indole-lyase. We have now prepared K256A, K256H, K256R, and E69D/K256R mutant TPLs to probe the role of Lys-256 in monovalent cation binding and activation. K256A and K256H TPLs have low activity (k(cat)/K(m) values of 0.01-0.1%), are not activated by monovalent cations, and do not exhibit fluorescence emission at 500 nm from the PLP cofactor. In contrast, K256R TPL has higher activity (k(cat)/K(m) about 10% of wild-type TPL), is activated by K(+), and exhibits fluorescence emission from the PLP cofactor. K256A, K256H, and K256R TPLs bind PLP somewhat weaker than wild-type TPL. E69D/K256R TPL was prepared to determine if the guanidine side chain could substitute for the monovalent cation. This mutant TPL has wild-type activity with S-Et-L-Cys or S-(o-nitrophenyl)-L-Cys but has no detectable activity with L-Tyr. E69D/K256R TPL is not activated by monovalent cations and does not show PLP fluorescence. In contrast to wild-type and other mutant TPLs, PLP binding to E69D/K256R is very slow, requiring several hours of incubation to obtain 1 mol of PLP per subunit. Thus, E69D/K256R TPL appears to have altered dynamics. All of the mutant TPLs react with inhibitors, L-Ala, L-Met, and L-Phe, to form equilibrating mixtures of external aldimine and quinonoid intermediates. Thus, Lys-256 is not the base which removes the alpha-proton during catalysis. The results show that the function of Lys-256 in TPL is in monovalent cation binding and activation.

To shed light on the mechanism of isotopic exchange of alpha-protons in amino acids catalyzed by pyridoxal phosphate (PLP)-dependent enzymes, we studied the kinetics of quinonoid intermediate formation for the reactions of tyrosine phenol-lyase with L-phenylalanine, L-methionine, and their alpha-deuterated analogues in D2O, and we compared the results with the rates of the isotopic exchange under the same conditions. We have found that, in the L-phenylalanine reaction, the internal return of the alpha-proton is operative, and allowing for its effect, the exchange rate is accounted for satisfactorily. Surprisingly, for the reaction with L-methionine, the enzymatic isotope exchange went much faster than might be predicted from the kinetic data for quinonoid intermediate formation. This result allows us to suggest the existence of an alternative, possibly concerted, mechanism of alpha-proton exchange.

Tyrosine phenol-lyase (TPL) and tryptophan indole-lyase (Trpase) catalyse the reversible hydrolytic cleavage of L-tyrosine or L-tryptophan to phenol or indole, respectively, and ammonium pyruvate. These enzymes are very similar in sequence and structure, but show strict specificity for their respective physiological substrates. We have mutated the active site residues of TPL (Thr(124), Arg(381), and Phe(448)) to those of Trpase and evaluated the effects of the mutations. Tyr(71) in Citrobacter freundii TPL, and Tyr(74) in E. coli Trpase, are essential for activity with both substrates. Mutation of Arg(381) of TPL to Ala, Ile, or Val (the corresponding residues in the active site of Trpase) results in a dramatic decrease in L-Tyr beta-elimination activity, with little effect on the activity of other substrates. Arg(381) may be the catalytic base with pK(a) of 8 seen in pH-dependent kinetic studies. T124D TPL has no measureable activity with L-Tyr or 3-F-L-Tyr as substrate, despite having high activity with SOPC. T124A TPL has very low but detectable activity, which is about 500-fold less than wild-type TPL, with L-Tyr and 3-F-L-Tyr. F448H TPL also has very low activity with L-Tyr. None of the mutant TPLs has any detectable activity with L-Trp as substrate. H463F Trpase also exhibits low activity with L-Trp, but retains high activity with other substrates. Thus, additional residues remote from the active site may be needed for substrate specificity. Both Trpase and TPL may react by a rare S(E)2-type mechanism.