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

Abstract

ABSTRACTBy selective enrichment, we isolated a bacterium that can use β-phenylalanine as a sole nitrogen source. It was identified by 16S rRNA gene sequencing as a strain ofVariovorax 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 inEscherichia coli. The recombinant enzyme was purified and showed a specific activity of 17.5 U mg−1for (S)-β-phenylalanine at 30°C and 33 U mg−1at the optimum temperature of 55°C. The β-specific aminotransferase exhibits a broad substrate range, acceptingortho-,meta-, andpara-substituted β-phenylalanine derivatives as amino donors and 2-oxoglutarate and pyruvate as amino acceptors. The enzyme is highly enantioselective toward (S)-β-phenylalanine (enantioselectivity [E], >100) and derivatives thereof with different substituents on the phenyl ring, allowing the kinetic resolution of various racemic β-amino acids to yield (R)-β-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 β-phenylalanine aminotransferase fromMesorhizobiumsp. strain LUK. The crystal structure was used to rationalize the stereo- and regioselectivity ofV. paradoxusaminotransferase and to define a sequence motif with which new aromatic β-amino acid-converting aminotransferases may be identified.