Phenylalanine Aminomutase Research Articles

Improving regioselectivity of phenylalanine aminomutase from Taxus chinensis by semi-rational mutagenesis for the biocatalytic amination of cinnamates.

The occupancy of the binding pocket by the substrate ultimately determines the outcome of enzyme catalysis. Previous engineering and substrate scope of phenylalanine aminomutase from Taxus chinensis (TcPAM) has generated valuable knowledge about the regioselectivity with biocatalytic potentials for the preparation of α- and β-phenylalanine and their derivatives. However, the significantly different regioselectivity during the amination of cinnamates by TcPAM is not fully understood. In this study, we take a reconstruction approach to change the whole binding pocket of TcPAM for probing the factors affecting the regioselectivity, resulting in variant C107S/Q319M/I431V reaching a 25.5-fold enhancement of the β/α product ratio toward trans-cinnamate acid. Furthermore, when substituted cinnamates were used as substrates, the regioselectivity was strongly correlated with various changes in the binding pocket, and value-added 2-Cl-α-Phe (100% α-selectivity) and 4-CH3-β-Phe (98% β-selectivity) were individually verified by the mutants L104A and Q319M at a preparative scale, exemplifying the application feasibility of our engineering strategy. The present study uncovered the cooperative connection between aromatic binding and carboxylate binding to affect the regioselectivity, which provides new insights into the determinants of the regioselectivity possessed by TcPAM and paves the way for its biocatalytic applications on phenylalanine derivatives.

Application of Priming Strategy for Enhanced Paclitaxel Biosynthesis in Taxus × Media Hairy Root Cultures

Despite huge progress in biotechnological approaches to paclitaxel production, Taxus spp. in vitro culture productivity still remains a challenge. This could be solved by developing a new strategy engaging mechanisms of the primed defence response joined with subsequent elicitation treatment to circumvent limitations in paclitaxel biosynthesis. The hairy roots were primed by preincubation with β-aminobutyric acid (BABA) for 24 h or 1 week, and then elicited with methyl jasmonate (MeJA) or a mixture of MeJA, sodium nitroprusside and L-phenylalanine (MIX). The effect of priming was evaluated on a molecular level by examination of the expression profiles of the four genes involved in paclitaxel biosynthesis, i.e., TXS (taxadiene synthase), BAPT (baccatin III: 3-amino, 3-phenylpropanoyltransferase), DBTNBT (3′-N-debenzoyl-2-deoxytaxol-N-benzoyltransferase) and PAM (phenylalanine aminomutase), as well as rolC (cytokinin-β-glucosidase), originated from the T-DNA of Agrobacterium rhizogenes. The maximum paclitaxel yield was achieved in cultures primed with BABA for 1 week and elicited with MIX (3179.9 ± 212 µg/g dry weight), which corresponded to the highest expression levels of TXS and BAPT genes. Although BABA itself induced the investigated gene expression over control level, it was not translated into paclitaxel production. Nevertheless, preincubation with BABA essentially affected paclitaxel yield, and the duration of BABA pretreatment seemed to have the most pronounced impact on its productivity.

Synthesis of (S)- and (R)-β-Tyrosine by Redesigned Phenylalanine Aminomutase

Phenylalanine aminomutase from Taxus chinensis (TchPAM) is employed in the biosynthesis of the widely used antitumor drug paclitaxel. TchPAM has received substantial attention due to its strict enantioselectivity towards (R)-β-phenylalanine, in contrast to the bacterial enzymes classified as EC 5.4.3.11 which are (S)-selective for this substrate. However, the understanding of the isomerization mechanism of the reorientation and rearrangement reactions in TchPAM might support and promote further research on expanding the scope of the substrate and thus the establishment of large-scale production of potential synthesis for drug development. Upon conservation analysis, computational simulation, and mutagenesis experiments, we report a mutant from TchPAM, which can catalyze the amination reaction of trans-p-hydroxycinnamic acid to (R)- and (S)-β-tyrosine. We propose a mechanism for the function of the highly conserved residues L179, N458, and Q459 in the active site of TchPAM. This work highlights the importance of the hydrophobic residues in the active site, including the residues L104, L108, and I431, for maintaining the strict enantioselectivity of TchPAM, and the importance of these residues for substrate specificity and activation by altering the substrate binding position or varying the location of neighboring residues. Furthermore, an explanation of (R)-selectivity in TchPAM is proposed based on the mutagenesis study of these hydrophobic residues. In summary, these studies support the future exploitation of the rational engineering of corresponding enzymes with MIO moiety (3,5-dihydro-5-methylidene-4H-imidazole-4-one) such as ammonia lyases and aminomutases of aromatic amino acids.

Computational-Designed Enzyme for β-Tyrosine Production in Lignin Valorization

Lignin is an underutilized sustainable source of aromatic compounds. To valorize the low-value lignin monomers, we proposed an efficient strategy, involving enzymatic conversion from trans-p-hydroxycinnamic acids to generate valued-added canonical and non-canonical aromatic amino acids. Among them, β-amino acids are recognized as building blocks for bioactive natural products and pharmaceutical ingredients due to their attractive antitumor properties. Using computational enzyme design, the (R)-β-selective phenylalanine aminomutase from Taxus chinensis (TchPAM) was successfully mutated to accept β-tyrosine as the substrate, as well as to generate the (R)-β-tyrosine with excellent enantiopurity (ee > 99%) as the unique product from trans-p-hydroxycinnamic acid. Moreover, the kinetic parameters were determined for the reaction of four Y424 enzyme variants with the synthesis of different phenylalanine and tyrosine enantiomers. In the ammonia elimination reaction of (R)-β-tyrosine, the variants Y424N and Y424C displayed a two-fold increased catalytic efficiency of the wild type. In this work, a binding pocket in the active site, including Y424, K427, I431, and E455, was examined for its influence on the β-enantioselectivity of this enzyme family. Combining the upstream lignin depolymerization and downstream production, a sustainable value chain based on lignin is enabled. In summary, we report a β-tyrosine synthesis process from a monolignol component, offering a new way for lignin valorization by biocatalyst modification.

Investigation into isomerization reaction of phenylalanine aminomutase from Pantoea agglomerans

Phenylalanine aminomutase (PaPAM) from Pantoea agglomerans is a member of the MIO (4-methylene-imidazol-5-one) family of enzymes, which isomerizes α-phenylalanine to β-phenylalanine, and could be used to synthesize unnatural β-arylalanine. However, the mechanism of isomerization reaction is not clear. To investigate the mechanism, the gene (pam), which encodes PaPAM, was first expressed in E.coli, and recombinant PaPAM was prepared using affinity chromatography. Then, 15N-(2S)-α-phenylalanine, (2S)-(3-2H2)-α-phenylalanine and (2S,3S)-[2,3-2H2]-α-phenylalanine were used as substrates to analyze the mechanism of isomerization reaction. The results of MS and NMR showed that the isomerization reaction was performed through the intramolecular exchange of NH2 with pro-3R hydrogen of α-phenylalanine. The PaPAM shuttles the α-NH2 of α-phenylalanine to β site to replace the pro-3R hydrogen. Simultaneously, the pro-3R hydrogen is shifted to α site to produce β-phenylalanine. Furthermore, a key residue, Phe at position 455 in the active site, was determined to control the exchange way using molecular docking and sequence alignment of MIO family enzymes. The results indicated that the key 455 Phe residue is involved in changing the binding orientation of the carboxyl group of the intermediate trans-cinnamic acid to control the NH2-H pair exchange.

Surface engineering of a Pantoea agglomerans-derived phenylalanine aminomutase for the improvement of (S)-β-phenylalanine biosynthesis

A Pantoea agglomerans-derived phenylalanine aminomutase (PaPAM) was engineered to improve the biocatalytic synthesis of (S)-β-phenylalanine, which is an important precursor of pharmaceuticals and peptidomimetics. A semi-rational design strategy based on a combination of surface-amino-acid engineering and the amino acid preference of the thermozyme was applied to counteract the enzyme trade-off between improving its activity and stability. The surface glycine, lysine and serine of PaPAM were mutated to alanine, arginine and alanine, respectively. A K340R mutant was screened with a 2.23-fold increased activity and 2.12-fold improved half-life at 50 °C over those of the wild-type PaPAM. These improvements resulted from the more stable enzymatic conformation as well as the more rigid inner loop in K340R. When tested in a whole-cell biocatalytic reaction, the (S)-β-phenylalanine volumetric productivity of K340R reached 0.47 g/L·h (1.4-fold greater than that of the wild-type PaPAM), and the conversion rate was improved by 17% compared to that of the wild-type PaPAM. The enzymatic properties of K340R and the resulting (S)-β-phenylalanine production are among the highest reported, and the results indicate that the described strategy is potent for engineering enzymatic stability and activity of PAM.

Understanding Which Residues of the Active Site and Loop Structure of a Tyrosine Aminomutase Define Its Mutase and Lyase Activities.

Site-directed mutations and substrate analogues were used to gain insights into the branch-point reaction of the 3,5-dihydro-5-methylidene-4 H-imidazol-4-one (MIO)-tyrosine aminomutase from Oryza sativa ( OsTAM). Exchanging the active residues of OsTAM (Y125C/N446K) for those in a phenylalanine aminomutase TcPAM altered its substrate specificity from tyrosine to phenylalanine. The aminomutase mechanism of OsTAM surprisingly changed almost exclusively to that of an ammonia lyase making cinnamic acid (>95%) over β-phenylalanine [Walter, T., et al. (2016) Biochemistry 55, 3497-3503]. We hypothesized that the missing electronics or sterics on the aryl ring of the phenylalanine substrate, compared with the sizable electron-donating hydroxyl of the natural tyrosine substrate, influenced the unexpected lyase reactivity of the OsTAM mutant. The double mutant was incubated with 16 α-phenylalanine substituent analogues of varying electronic strengths and sterics. The mutant converted each analogue principally to its acrylate with ∼50% conversion of the p-Br substrate, making only a small amount of the β-amino acid. The inner loop structure over the entrance to the active site was also mutated to assess how the lyase and mutase activities are affected. An OsTAM loop mutant, matching the loop residues of TcPAM, still chiefly made >95% of the acrylate from each substrate. A combined active site:loop mutant was most reactive but remained a lyase, making 10-fold more acrylates than other mutants did. While mutations within the active site changed the substrate specificity of OsTAM, continued exploration is needed to fully understand the interplay among the inner loop, the substrate, and the active site in defining the mutase and lyase activities.

One step synthesis of unnatural β-arylalanines using mutant phenylalanine aminomutase from Taxus chinensis with high β-regioselectivity.

Phenylalanine aminomutase (TcPAM) from Taxus chinensis catalyzes the regioselective hydroamination of trans-cinnamic acid (t-CA) to yield β-phe. However, the final product mixture consists of both α- and β-phe owing to low regioselectivity, which is still a challenge to synthesize highly pure β-phe. Therefore, a modified TcPAM with high β-selectivity is expected. Based on the catalytic mechanism and structure, two amino acid residues (Asn458 and Leu108) in active sites were identified as the key residues for controlling the regioselective hydroamination of t-CA and as promising candidates for mutagenesis to enhance β-selectivity and decrease α-selectivity. The Asn458 and Leu108 residues were mutated to yield variant TcPAM-Asn458Phe/Leu108Glu, and the β-selectivity was approximately 5.2-fold higher than that of wild-type TcPAM, while α-selectivity decreased to 68%, and the percentage of β-phe in the product mixture increased from 42% to 83%. In addition, the mutant was applied to synthesize β-arylalanines using substituent t-CA as a substrate. The regioselectivity was also affected by the substituent groups at the phenyl ring of t-CA with respect to their electronic properties and position, and the 4-methoxy and methyl substituent t-CA were transferred into β-arylalanines. The conversion rate also exceeded 90%. In summary, the engineered TcPAM proved to be useful for one-step asymmetric amination of t-CA and its derivatives to synthesize highly pure β-arylalanines.

Kinetic Resolution of Aromatic β‐Amino Acids Using a Combination of Phenylalanine Ammonia Lyase and Aminomutase Biocatalysts

AbstractAn enzymatic strategy for the preparation of (R)‐β‐arylalanines employing phenylalanine aminomutase and ammonia lyase (PAM and PAL) enzymes has been demonstrated. Candidate PAMs with the desired (S)‐selectivity from Streptomyces maritimus (EncP) and Bacillus sp. (PabH) were identified via sequence analysis using a well‐studied template sequence. The newly discovered PabH could be linked to the first ever proposed biosynthesis of pyloricidin‐like secondary metabolites and was shown to display better β‐lyase activity in many cases. In spite of this, a method combining the higher conversion of EncP with a strict α‐lyase from Anabaena variabilis (AvPAL) was found to be more amenable, allowing kinetic resolution of five racemic substrates and a preparative‐scale reaction with >98% (R) enantiomeric excess. This work represents an improved and enantiocomplementary method to existing biocatalytic strategies, allowing simple product separation and modular telescopic combination with a preceding chemical step using an achiral aldehyde as starting material.magnified image

Mutation of Aryl Binding Pocket Residues Results in an Unexpected Activity Switch in an Oryza sativa Tyrosine Aminomutase.

A 3,5-dihydro-5-methylidine-4H-imidazol-4-one (MIO)-dependent tyrosine aminomutase (TAM) isolated from the rice plant Oryza sativa (OsTAM) makes β-tyrosine (75%) and p-coumarate (25%) from α-tyrosine. OsTAM is the first TAM to have, although slight, native phenylalanine aminomutase (PAM) activity (3% relative to TAM activity). The active sites of OsTAM and a TcPAM from Taxus plants differ by only two residues (Y125 and N446 of OsTAM vs C107 and K427 of TcPAM) positioned similarly near the aryl ring of their substrates. The kinetic parameters and substrate selectivity were measured for OsTAM single mutants Y125C and N446K OsTAM and double mutant Y125C/N446K OsTAM. Compared with OsTAM, each single mutant was slower at converting α-tyrosine to its β-isomer and p-coumarate; the double mutant did not produce any detectable product. Each mutant bound α-phenylalanine ∼9-fold better than did OsTAM, suggesting that the mutations made the catalysts more selective for phenylalanine. The total turnover rate (kcatβ-Ph...

A Tyrosine Aminomutase from Rice (Oryza sativa) Isomerizes (S)-α- to (R)-β-Tyrosine with Unique High Enantioselectivity and Retention of Configuration.

A recently discovered 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO)-dependent tyrosine aminomutase (OsTAM) from rice [Yan, J., et al. (2015) Plant Cell 27, 1265] converts (S)-α-tyrosine to a mixture of (R)- and (S)-β-tyrosines, with high (94%) enantiomeric excess, which does not change with pH, like it does for two bacterial TAMs. The K(M) of 490 μM and the k(cat) of 0.005 s(-1) are similar for other TAM enzymes. OsTAM is unique and also catalyzes (R)-β- from (S)-α-phenylalanine. OsTAM principally retains the configuration at the reactive C(α) and C(β) centers during catalysis much like the phenylalanine aminomutase on the Taxol biosynthetic pathway in Taxus plants.

Whole-cell biocatalytic production of variously substituted β-aryl- and β-heteroaryl-β-amino acids

Biologically-active β-peptides and pharmaceuticals that contain key β-amino acids are emerging as target therapeutics; thus, synthetic strategies to make substituted, enantiopure β-amino acids are increasing. Here, we use whole-cell Escherichia coli (OD600 ∼35) engineered to express a Pantoea agglomerans phenylalanine aminomutase (PaPAM) biocatalyst. In either 5mL, 100mL, or 1L of M9 minimal medium containing α-phenylalanine (20mM), the cells produced ∼1.4mgmL−1 of β-phenylalanine in each volume. Representative pilot-scale 5-mL cultures, fermentation reactions converted 18 variously substituted α-arylalanines to their (S)-β-aryl-β-amino acids in vivo and were not toxic to cells at mid- to late-stage growth. The β-aryl-β-amino acids made ranged from 0.043mg (p-nitro-β-phenylalanine, 4% converted yield) to 1.2mg (m-bromo-β-phenylalanine, 96% converted yield) over 6h in 5mL. The substituted β-amino acids made herein can be used in redox and Stille-coupling reactions to make synthetic building blocks, or as bioisosteres in drug design.

Ironing out their differences: dissecting the structural determinants of a phenylalanine aminomutase and ammonia lyase.

Deciphering the structural features that functionally separate ammonia lyases from aminomutases is of interest because it may allow for the engineering of more efficient aminomutases for the synthesis of unnatural amino acids (e.g., β-amino acids). However, this has proved to be a major challenge that involves understanding the factors that influence their activity and regioselectivity differences. Herein, we report evidence of a structural determinant that dictates the activity differences between a phenylalanine ammonia lyase (PAL) and aminomutase (PAM). An inner loop region that closes the active sites of both PAM and PAL was mutated within PAM (PAM residues 77-97) in a stepwise approach to study the effects when the equivalent residue(s) found in the PAL loop were introduced into the PAM loop. Almost all of the single loop mutations triggered a lyase phenotype in PAM. Experimental and computational evidence suggest that the induced lyase features result from inner loop mobility enhancements, which are possibly caused by a 310-helix cluster, flanking α-helices, and hydrophobic interactions. These findings pinpoint the inner loop as a structural determinant of the lyase and mutase activities of PAM.

Ring-Substituted α-Arylalanines for Probing Substituent Effects on the Isomerization Reaction Catalyzed by an Aminomutase

β-Amino acids are emerging as an important class of compounds in medicinal chemistry. β-Aryl-β-alanines show antiepileptogenesis activity, while others have been used to synthesize antibiotic β-peptides. To assess the utility of a methylidene imidazolone-dependent Pantoea agglomerans phenylalanine aminomutase (PaPAM) for making non-natural β-amino acids, we surveyed the substrate specificity of PaPAM with several commercially available (S)-arylalanine substrates. Here, we report the Michaelis–Menten parameters and catalytic efficiency of PaPAM for each substrate. Compared to phenylalanine, substrates containing substituents that were either electron-withdrawing or -donating through resonance or inductive effects affected the kcat of PaPAM. Generally, the turnover and catalytic efficiency of PaPAM for the meta-isomers were better than for the corresponding para- and ortho-isomers, with some exceptions. PaPAM principally synthesizes the β-amino acids at >90% and the cinnamate byproducts at <10% for 11 of the 19 productive substrates. The yield from other substrates was 14–65% of the cinnamate analogue. Further, to explain the determinants of substrate selectivity of PaPAM, a series of substrates with substituents on the aryl ring were docked into the crystal structure of the active site. Induced fit of the protein to accommodate different substituents was modeled computationally by SLIDE docking and Szybki energy minimization. The results provide insights into the roles of substrate orientation and conformational flexibility in turnover and indicate which terms of the interaction energy account for the experimentally observed KM values, which largely determine catalytic efficiency. Substrate selectivity of PaPAM is significantly influenced by steric barriers created by specific active-site residue interactions with the substituted aryl portion of the substrate.

From Aminomutases to Ammonia Lyases: A Protein Engineering Study

Ammonia lyases and aminomutases are emerging as important enzymatic systems, not only in green synthetic routes to chiral amines, but also as potential target for enzyme therapeutic for treating diseases such as phenylketonuria and cancer (1). On the other hand, β-amino acids harbor many applications in their free form and as building blocks of bioactive compounds (2, 3). Although, these eco-friendly biocatalytic routes have been extensively explored, they are far from optimal. The aim of this work is to engineer a phenylalanine aminomutase (PAM) to acquire lyase properties for the efficient production of enantio-pure β-Phe (key component of taxol (2)). Thus, this study was guided by molecular modeling techniques to decipher which structural components functionally separate PAM and the phenylalanine ammonia lyase (PAL). Despite the great structural similarity of the active site of these enzymes, PAL is α-selective with much faster deamination rates relative to PAM, which exhibits 50% α- and β-regioselectivity (1, 4). Recent studies have implicated loop regions as key structural determinants between PAM and PAL (5). Here, we report novel insight into the implications of the active-site loop residues of PAM, which influence mutase/lyase activity. Several mutants were proposed, cloned, expressed and characterized. Overall, this enzyme engineering work represents the first successful attempt to convert a PAM to a PAL through strict mutase-to-lyase residue mutations. Such a breakthrough may guide future investigations into the functional determinants of these enzymes and possibly foster the engineering of faster PAM variants used for the efficient synthesis of β-Phe. 1. Heberling, M.M., et al. 2013. Current Opinion in Chemical Biology. 2. Malik, S., et al. 2011. Process Biochemistry. 3. Liljeblad, A. et al. 2006. Tetrahedron. 4. Wu, B., et al. 2011. Angew. Chem. Int. Ed. 5. Bartsch, S., et al. 2013. ChemCatChem.

Insight into the mechanism of aminomutase reaction: A case study of phenylalanine aminomutase by computational approach

The Taxus canadensis phenylalanine aminomutase (TcPAM) catalyze the isomerization of (S)-α-phenylalanine to the (R)-β-isomer. The active site of TcPAM contains the signature 5-methylene-3,5-dihydroimidazol-4-one (MIO) prosthesis, observed in the ammonia lyase class of enzymes. Up to now, there are two plausible mechanisms for these MIO-dependent enzymes, i.e., the amino-MIO adduct mechanism and the Friedel–Crafts-type reaction mechanism. In response to this mechanistic uncertainty, the phenylalanine aminomutase mechanism was investigated by using density functional methods. The calculation results indicate that: (1) the reaction prefers the amino-MIO adduct mechanism where the 2,3-amine shift process contains six elementary steps; (2) the ammonia elimination step proceeds through an E2 mechanism; (3) a single C1Cα bond rotation of 180° in the cinnamate skeleton occurs in the active site prior to the rebinding of NH2 group to the cinnamate. This can be used to explain the stereochemistry of the TcPAM reaction product which is contrary to those of the PaPAM and SgTAM enzymes. Based on these calculations, the roles of important residues in the active site were also elucidated.

Molecular Cloning and Characterization of the Phenylalanine Aminomutase Gene From Taxus baccata L.

Background: Taxol is one of the most important anti-cancer drugs, which is obtained from yew trees (Taxus sp.). T+he fist step in side chain assembly of taxol is catalyzed by phenylalanine aminomutase, which converts α-phenylalanine to β-phenylalanine. Objectives: In this study, for the fist time, we report on the cloning, preliminary expression and characterization of a full-length gene and cDNA encoding phenylalanine aminomutase from Taxus baccata L. Materials and Methods: Comparison of the full-length gene with other ones identifid from the Taxus species showed high similarity, particularly with Taxus x media. Results: The results showed that the expression level of this gene in Taxus baccata is very low and therefore this enzymatic step could be a rate limiting step in the taxol biosynthesis pathway. Successful amplifiation of the cDNA was only obtained from RNA samples isolated from methyl jasmonate elicited suspension cells of Taxus baccata. The cloned cDNA contained a 2064 bp open reading frame encoding a protein composed of 687 amino acids. Sequence comparison analysis revealed that the gene is very similar (98 - 99 %) with respect to the nucleotide and amino acid sequences in diffrent Taxus species and also share the signature active site motif (175ASG177). Conclusions: The predicted structure of TbPAM was analyzed using bioinformatic tools. The results indicated that the protein has similar overall folding to tyrosine aminomutase.

Redesign of a Phenylalanine Aminomutase into a Phenylalanine Ammonia Lyase

AbstractAn aminomutase, naturally catalyzing the interconversion of (S)‐α‐phenylalanine and (R)‐β‐phenylalanine, was converted into an ammonia lyase catalyzing the nonoxidative deamination of phenylalanine to cinnamic acid by a rational single‐point mutation. It could be shown by crystal structures and kinetic data that the flexibility of the lid that covers the active site decides whether the enzyme acts as a lyase or a mutase. An Arg92Ser mutation destabilized the closed conformation of the lid structure and converted the mutase into a lyase that exhibited up to 44‐fold increased reaction rates in the enantioselective deamination of (R)‐β‐phenylalanine. In addition, the amination rates of cinnamic acid yielding optically pure (S)‐α‐ and (R)‐β‐phenylalanine were doubled. The applicability of the mutant enzyme for kinetic resolution and asymmetric amination could be shown by biocatalysis on a preparative scale.

Assessing the Deamination Rate of a Covalent Aminomutase Adduct by Burst Phase Analysis

Burst-phase kinetic analysis was used to evaluate the deamination rate of the aminated-methylidene imidazolone (NH(2)-MIO) adduct of a Taxus phenylalanine aminomutase. The kinetic parameters were interrogated by a non-natural substrate (S)-styryl-α-alanine that yielded a chromophoric styrylacrylate product upon deamination by the aminomutase. Transient inactivation of the enzyme by the NH(2)-MIO adduct intermediate resulted in an initial burst of product, with reactivation by deamination of the adduct. This study validated the rate constants of a kinetic model demonstrating that the NH(2)-MIO adduct and cinnamate intermediate are sufficiently retained to catalyze the natural α- to β-phenylalanine isomerization.

Thermal Bifunctionality of Bacterial Phenylalanine Aminomutase and Ammonia Lyase Enzymes

Enzymatic thermal switch: The bacterial 4-methylideneimidazol-5-one (MIO) dependent enzymes AdmH and EncP are shown to display remarkable thermal bifunctionality: they act as mutases (blue graph) at lower temperatures but with lyase (red graph) activity predominant at higher temperatures. This temperature-dependent switch in enzyme class also explains how these two similar enzymes can fulfill different catalytic functions in secondary metabolisms.