Engineering of an enantioselective tyrosine aminomutase by mutation of a single active site residue in phenylalanine aminomutase

Wu, Bian; Szymański, Wiktor; Wijma, Hein J.; Crismaru, Ciprian G.; Wildeman, S. De; Poelarends, Gerrit J.; Feringa, Ben L.; Janssen, Dick B.

doi:10.1039/c0cc02768e

Cited by 23 publications

(22 citation statements)

References 24 publications

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“…The main difference is the intensively investigated substrate selectivity switch3e Cys107 in PAM (Phe137 in PAL) that was used to alter the substrate specificity of mutases and lyases 3d. e, 5, 7 In addition, the crystal structures of both enzymes (PAM, PDB codes: 3NZ48 and 2YII;3a and PAL, PDB code: 1W279) are very similar (RMSD for 1197 C α ‐atoms is 1.2 Å, 46 % sequence identity)10 (Figure 1 a). Although the active sites of both enzymes are nearly identical, PAL does not have aminomutase activity.…”

Section: Resultsmentioning

confidence: 99%

Redesign of a Phenylalanine Aminomutase into a Phenylalanine Ammonia Lyase

et al. 2013

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An 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.

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Section: Resultsmentioning

confidence: 99%

Redesign of a Phenylalanine Aminomutase into a Phenylalanine Ammonia Lyase

et al. 2013

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“…The strong similarity of L-phenylalanine and L-tyrosine 2,3-aminomutases (PAM (40)(41)(42)(43) and TAM (44,45), respectively) to the ammonia-lyase family is indicated by the presence of unusual catalytic MIO in these aminomutases and also by the fact that both PAM (46) and TAM (47) have ammonia-lyase activity.…”

Section: Introductionmentioning

confidence: 97%

Preparation of Unnatural Amino Acids with Ammonia-Lyases and 2,3-Aminomutases

Poppe

Paizs

Kovács

et al. 2011

Methods in Molecular Biology

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Ammonia-lyases catalyze a wide range of processes leading to α,β-unsaturated compounds by elimination of ammonia. In this chapter, ammonia-lyases are reviewed with major emphasis on their synthetic applications in stereoselective preparation of unnatural amino acids. Besides the synthesis of various unnatural α-amino acids with the aid of phenylalanine ammonia-lyases (PALs) utilizing the 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) prosthetic groups, the biotransformations leading to various unnatural β-amino acids with phenylalanine 2,3-aminomutases using the same catalytic MIO prosthetic group are discussed. Cloning, production, purification, and biotransformation protocols for PAL are described in detail.

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“…These latter enzymes catalyze the deamination of the corresponding l -amino acids (HAL: L-His; TAL: L-Tyr) [18], [19]. The family of ammonia-lyases show a strong similarity to the family of aminomutase enzymes ( l -phenylalanine and l -tyrosine 2,3-aminomutases, PAM [20]–[23] and TAM [24], [25], respectively) as indicated by presence of the unusual catalytic MIO moiety in both ammonia-lyases and these aminomutases and also by the fact that both PAM [20] and TAM [26] have ammonia-lyase activity. The amino acid residues involved in formation of the MIO moiety constitute a strictly conserved tripeptide of alanine, serine and glycine (ASG, see Figure 1).…”

Section: Introductionmentioning

confidence: 99%

Expression and Properties of the Highly Alkalophilic Phenylalanine Ammonia-Lyase of Thermophilic Rubrobacter xylanophilus

et al. 2014

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The sequence of a phenylalanine ammonia-lyase (PAL; EC: 4.3.1.24) of the thermophilic and radiotolerant bacterium Rubrobacter xylanophilus (RxPAL) was identified by screening the genomes of bacteria for members of the phenylalanine ammonia-lyase family. A synthetic gene encoding the RxPAL protein was cloned and overexpressed in Escherichia coli TOP 10 in a soluble form with an N-terminal His6-tag and the recombinant RxPAL protein was purified by Ni-NTA affinity chromatography. The activity assay of RxPAL with l-phenylalanine at various pH values exhibited a local maximum at pH 8.5 and a global maximum at pH 11.5. Circular dichroism (CD) studies showed that RxPAL is associated with an extensive α-helical character (far UV CD) and two distinctive near-UV CD peaks. These structural characteristics were well preserved up to pH 11.0. The extremely high pH optimum of RxPAL can be rationalized by a three-dimensional homology model indicating possible disulfide bridges, extensive salt-bridge formation and an excess of negative electrostatic potential on the surface. Due to these properties, RxPAL may be a candidate as biocatalyst in synthetic biotransformations leading to unnatural l- or d-amino acids or as therapeutic enzyme in treatment of phenylketonuria or leukemia.

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Engineering of an enantioselective tyrosine aminomutase by mutation of a single active site residue in phenylalanine aminomutase

Cited by 23 publications

References 24 publications

Redesign of a Phenylalanine Aminomutase into a Phenylalanine Ammonia Lyase

Redesign of a Phenylalanine Aminomutase into a Phenylalanine Ammonia Lyase

Preparation of Unnatural Amino Acids with Ammonia-Lyases and 2,3-Aminomutases

Expression and Properties of the Highly Alkalophilic Phenylalanine Ammonia-Lyase of Thermophilic Rubrobacter xylanophilus

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