Journal of Biotechnology 128 (2007) 408–411 Short communication Phenylalanine dehydrogenase mutants: Efficient biocatalysts for synthesis of non-natural phenylalanine derivatives Francesca Paradisi a , Stuart Collins b , Anita R. Maguire b , Paul C. Engel a,∗ a UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland b Department of Chemistry, Analytical and Biological Chemistry Research Facility, University College, Cork, Cork, Ireland Received 11 May 2006; received in revised form 2 August 2006; accepted 15 August 2006 Abstract Wild-type phenylalanine dehydrogenase from Bacillus sphaericus, and three mutants N145A, N145V and N145L, are used with a coenzyme recycling system to synthesise l-phenylalanine and three non-natural amino acids (p-F-phenylalanine, p- MeO-phenylalanine and p-CF 3 -phenylalanine) on a millimole scale. A range of reaction conditions are investigated. The kinetic parameters of WT PheDH and N145A towards p-CF 3 -phenylpyruvate are compared, emphasising the value of protein engineering in creating improved biocatalysts. © 2006 Elsevier B.V. All rights reserved. Keywords: Non-natural amino acids; Phenylalanine dehydrogenase; Biocatalysis; Cofactor recycling; Chiral synthesis; Site-directed mutagenesis 1. Introduction Phenylalanine dehydrogenase (PheDH) from Bacil- lus sphaericus performs the reductive amination of phenylpyruvate, producing phenylalanine, but also accepts several other 2-oxo acids, yielding the cor- responding l-amino acids (Asano et al., 1990). We recently (Busca et al., 2004) reported that genetically engineered PheDHs, with extended substrate speci- ficity, are valuable catalysts for creating non-natural amino acids, increasingly in demand as pharmaceu- ∗ Corresponding author. Tel.: +353 1 716 6764; fax: +353 1 283 7211. E-mail address: paul.engel@ucd.ie (P.C. Engel). tical synthons, from synthetic 2-oxo acids. Critically, the mutants retained enantioselectivity, generating only the l-amino acid, and their activity in forming various non-natural amino acids was greatly enhanced relative to the WT enzyme. Meiwes et al. (1997) have reported similar use of an engineered transaminase in synthesis of non-natural amino acids. Stoichiometric consumption of costly NAD + / NADH in dehydrogenase-catalysed syntheses is fre- quently avoided by recycling with formate dehydroge- nase (FDH) or alcohol dehydrogenase (ADH). Asano et al. (1990) used PheDH with FDH to synthesise l-Phe. In our studies ADH offers advantages: with non-natural substrates, insolubility can be a problem, and ethanol serves both as a substrate for ADH and as a co-solvent. 0168-1656/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2006.08.008