Biochemical properties and crystal structure of a ?-phenylalanine aminotransferase from Variovorax paradoxus.

ABSTRACT: 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.

SUBMITTER: Crismaru CG 

PROVIDER: S-EPMC3536118 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature