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

Ciprian G. Crismaru, Gjalt G. Wybenga, Wiktor Szymanski, Hein J. Wijma, Bian Wu, Sebastian Bartsch, Stefaan de Wildeman, Gerrit J. Poelarends, Ben L. Feringa, Bauke Dijkstra, Dick B. Janssen*

*Bijbehorende auteur voor dit werk

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Samenvatting

By selective enrichment, we isolated a bacterium that can use beta-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)-beta-phenylalanine at 30 degrees C and 33 U mg(-1) at the optimum temperature of 55 degrees C. The beta-specific aminotransferase exhibits a broad substrate range, accepting ortho-, meta-, and para-substituted beta-phenylalanine derivatives as amino donors and 2-oxoglutarate and pyruvate as amino acceptors. The enzyme is highly enantioselective toward (S)-beta-phenylalanine (enantioselectivity [E], > 100) and derivatives thereof with different substituents on the phenyl ring, allowing the kinetic resolution of various racemic beta-amino acids to yield (R)-beta-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 beta-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 beta-amino acid-converting aminotransferases may be identified.

Originele taal-2English
Pagina's (van-tot)185-195
Aantal pagina's11
TijdschriftApplied and environmental microbiology
Volume79
Nummer van het tijdschrift1
DOI's
StatusPublished - jan-2013

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