4AO9

Biochemical properties and crystal structure of a novel beta- phenylalanine aminotransferase from Variovorax paradoxus

Summary for 4AO9

• Entry DOI: 10.2210/pdb4ao9/pdb
• Descriptor: BETA-PHENYLALANINE AMINOTRANSFERASE, PYRIDOXAL-5'-PHOSPHATE, GLYCEROL, ... (4 entities in total)
• Functional Keywords: transferase
• Biological source: VARIOVORAX PARADOXUS
• Total number of polymer chains: 2
• Total formula weight: 97856.21

Authors

Crismaru, C.G.,Wybenga, G.G.,Szymanski, W.,Wijma, H.J.,Wu, B.,deWildeman, S.,Poelarends, G.J.,Dijkstra, B.W.,Janssen, D.B. (deposition date: 2012-03-25, release date: 2012-10-24, Last modification date: 2024-05-01)

Primary citation

Crismaru, C.G.,Wybenga, G.G.,Szymanski, W.,Wijma, H.J.,Wu, B.,Dewildeman, S.,Poelarends, G.J.,Dijkstra, B.W.,Janssen, D.B.
Biochemical Properties and Crystal Structure of a Novel Beta-Phenylalanine Aminotransferase from Variovorax Paradoxus
Appl.Environ.Microbiol., 79:185-, 2013

PubMed 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.

PubMed: 23087034
DOI: 10.1128/AEM.02525-12

Experimental method

X-RAY DIFFRACTION (1.5 Å)