Biochemical Properties and Crystal Structure of a β-Phenylalanine Aminotransferase from Variovorax paradoxus

Abstract

e By selective enrichment, we isolated a bacterium that can use ␤-phenylalanine as a sole nitrogen source. It was identified by 16S rRNA gene sequencing as a strain of Variovorax paradoxus. Enzyme assays revealed an aminotransferase activity. Partial genome sequencing and screening of a cosmid DNA library resulted in the identification of a 1,302-bp aminotransferase gene, which encodes a 46,416-Da protein. The gene was cloned and overexpressed in Escherichia coli. The recombinant enzyme was purified and showed a specific activity of 17.5 U mg ؊1 for (S)-␤-phenylalanine at 30°C and 33 U mg ؊1 at the optimum temperature of 55°C. The ␤-specific aminotransferase exhibits a broad substrate range, accepting ortho-, meta-, and para-substituted ␤-phenylalanine derivatives as amino donors and 2-oxoglutarate and pyruvate as amino acceptors. The enzyme is highly enantioselective toward (S)-␤-phenylalanine (enantioselectivity [E], >100) and derivatives thereof with different substituents on the phenyl ring, allowing the kinetic resolution of various racemic ␤-amino acids to yield (R)-␤-amino acids with >95% enantiomeric excess (ee). The crystal structures of the holoenzyme and of the enzyme in complex with the inhibitor 2-aminooxyacetate revealed structural similarity to the ␤-phenylalanine aminotransferase from Mesorhizobium sp. strain LUK. The crystal structure was used to rationalize the stereo-and regioselectivity of V. paradoxus aminotransferase and to define a sequence motif with which new aromatic ␤-amino acid-converting aminotransferases may be identified.

Biochemical properties of a Pseudomonas aminotransferase involved in caprolactam metabolism

“…Three β‐amino acids were tested, but none was converted by any of the enzymes (Table ). This clearly distinguishes these aminotransferases from the homologous PLP fold type I β‐amino acid aminotransferases discovered in bacterial cultures enriched with β‐Phe as growth substrate . Likewise, no good conversion of proteinogenic amino acids was found, in case of Cv AT and Vf AT agreement with Kaulman et al .…”

“…Aminotransferases often belong to fold type I or fold type IV PLP enzymes . Examples of well‐studied fold type I aminotransferases are l ‐aspartate ATs , branched l ‐amino acid ATs, β‐amino acid ATs and the ω‐aminotransferases (ωATs) from Vibrio fluvialis, Chromobacterium violaceum , Paracoccus denitrificans , and Ochrobactrum anthropi . Fold type IV enzymes include d ‐amino acid ATs and ( R )‐amine selective ωATs .…”

Improvement in the Thermostability of a β‐Amino Acid Converting ω‐Transaminase by Using FoldX

“…Besides this, β‐PA and derivatives thereof can also be utilized in β‐peptides, as inhibitor for peptidase IV and in drug‐delivery systems . The β‐PA‐converting ω‐TA from S. thermophiles shows only a low activity of 3.3 U mg −1 compared to the ω‐TA from Variovorax paradoxus with a specific activity of 17.5 U mg −1 at 37 °C . Thus, the transaminase from V. paradoxus is an interesting candidate for enzyme engineering taking into account its high enzymatic activity and lack of data concerning thermostabilization.…”

One‐step purification and immobilization of a β‐amino acid aminotransferase using magnetic (M‐PVA) beads

“…At 40°C the activity of the immobilized enzyme decreased 20%. The activities were also compared to the activity of the free enzyme from Crismaru et al [33]. For the examination of the thermal stability the immobilized and crude cell extract were incubated for 30 min at certain temperature.…”

“…In the study presented here, kinetic resolution of enantiopure β-amino acids was conducted via an immobilized His-tagged β-amino acid AT from Variovorax paradoxus characterized by Crismaru et al [33].…”