title: Biochemical properties and crystal structure of a β-phenylalanine aminotransferase from Variovorax paradoxus.
abstract: By 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 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)-β-phenylalanine at 30°C and 33 U mg(-1) at the optimum temperature of 55°C. The β-specific aminotransferase exhibits a broad substrate range, accepting ortho-, meta-, and para-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 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 β-amino acid-converting aminotransferases may be identified.
authors.affiliation: Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.
authors.forename: Ciprian G
authors.initials: CG
authors.lastname: Crismaru
authors.forename: Gjalt G
authors.initials: GG
authors.lastname: Wybenga
authors.forename: Wiktor
authors.initials: W
authors.lastname: Szymanski
authors.forename: Hein J
authors.initials: HJ
authors.lastname: Wijma
authors.forename: Bian
authors.initials: B
authors.lastname: Wu
authors.forename: Sebastian
authors.initials: S
authors.lastname: Bartsch
authors.forename: Stefaan
authors.initials: S
authors.lastname: de Wildeman
authors.forename: Gerrit J
authors.initials: GJ
authors.lastname: Poelarends
authors.forename: Ben L
authors.initials: BL
authors.lastname: Feringa
authors.forename: Bauke W
authors.initials: BW
authors.lastname: Dijkstra
authors.forename: Dick B
authors.initials: DB
authors.lastname: Janssen
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D042621:Comamonadaceae / Q000737:chemistry / Q000201:enzymology* / Q000302:isolation & purification / Q000378:metabolism; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010649:Phenylalanine / Q000378:metabolism*; D011487:Protein Conformation; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature; D000637:Transaminases / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D011994:Recombinant Proteins; D010649:Phenylalanine; D000637:Transaminases; C022340:phenylalanine aminotransferase