4aoa

Publication Abstract from PubMed

By selective enrichment we isolated a bacterium that can use beta-phenylalanine as 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 towards (S)-beta-phenylalanine (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% ee. The crystal structures of the holo-enzyme and of the enzyme in complex with the inhibitor 2-aminooxyacetate revealed structural similarity to the beta-phenylalanine aminotransferase from Mesorhizobium sp. 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.

Biochemical properties and crystal structure of a beta-phenylalanine aminotransferase from Variovorax paradoxus.,Crismaru CG, Wybenga GG, Szymanski W, Wijma HJ, Wu B, Bartsch S, de Wildeman S, Poelarends GJ, Feringa BL, Dijkstra BW, Janssen DB Appl Environ Microbiol. 2012 Oct 19. PMID:23087034^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.