Two Enantiocomplementary Ephedrine Dehydrogenases from Arthrobacter sp. TS-15 with Broad Substrate Specificity

The recently identified pseudoephedrine and ephedrine dehydrogenases from Arthrobacter sp. TS-15, PseDH and EDH, are NADH-dependent members of the oxidoreductase superfamily of short-chain dehydrogenases/reductases (SDRs). They are specific for the enantioselective oxidation of (+)-(S) N-(pseudo)ephedrine and (-)-(R) N-(pseudo)ephedrine, respectively. Anti-Prelog stereospecific PseDH and Prelog-specific EDH catalyse the regio- and enantiospecific reduction of 1-phenyl-1,2-propanedione to (S)-phenylacetylcarbinol and (R)-phenylacetylcarbinol with full conversion and enantiomeric excess of >99%. Moreover, they perform the reduction of a wide range of aryl aliphatic carbonyl compounds, including keto amines, ketoesters and haloketones, to the corresponding enantiopure alcohols. The highest stability of PseDH and EDH was determined to be at a pH range of 6.0-8.0 and 7.5-8.5, respectively. PseDH was more stable than EDH at 25 °C, with half-lives of 279 h and 38 h respectively. However, EDH is more stable at 40 °C with a two-fold greater half-life than at 25 °C. The crystal structure of the PseDH-NAD+ complex, refined to a resolution of 1.83 Å, revealed a tetrameric structure, which was confirmed by solution studies. A model of the active site in complex with NAD+ and 1-phenyl-1,2-propanedione suggested key roles for S143 and W152 in recognition of the substrate and positioning for the reduction reaction. The wide substrate spectrum of these dehydrogenases, combined with their regio- and enantioselectivity, suggests high potential for the industrial production of valuable chiral compounds.