Unusual Mechanism for an Aminomutase Rearrangement:  Retention of Configuration at the Migration Termini

Abstract

The phenylalanine aminomutase from Taxus catalyzes the vicinal exchange of the amino group and the pro-3S hydrogen of (2S)-alpha-phenylalanine to make (3R)-beta-phenylalanine. While the migration of the amino group from C2 of the substrate to C3 of the product is already known to proceed intramolecularly with retention of configuration, the stereochemistry of the hydrogen transfer remained unknown, until now. The chemical shifts of the prochiral hydrogens of authentic (3R)-beta-phenylalanine were established by 1H NMR, and the configuration of each hydrogen was assigned by 2H NMR analysis of a racemic mixture of [2,3-2H2]-(2S,3R)- and (2R,3S)-beta-phenylalanines synthesized via syn addition of deuterium gas with palladium catalyst to stereospecifically reduce the double bond of an N-acetyl enamine. After the aminomutase was incubated with [3,3-2H2]-(2S)-alpha-phenylalanine, the derived deuterium-labeled beta-diastereoisomer product, derivatized as the N-acetyl methyl ester, was analyzed by 2H NMR, which revealed that the mutase shuttles the pro-3S hydrogen to C2 of the beta-isomer product (designated 2S,3R) with retention of configuration. Retention of configuration at both reaction termini is unique among all aminomutase mechanisms examined so far. Furthermore, the dynamics of the Cbeta-H bond of the substrate were measured in a competitive experiment with deuterium-labeled substrate to calculate a primary kinetic isotope effect on Vmax/KM of 2.0 +/- 0.2, indicating that C-H bond cleavage is likely rate limiting. Isotope exchange data indicate that the migratory deuterium of [2H8]-(2S)-alpha-phenylalanine, at saturation, dynamically exchanges up to 75%, with protons from the solvent during the reaction after the first 10% of product is formed. The calculated equilibrium constant of 1.1 indicates that the beta-isomer was slightly favored relative to the alpha-isomer at 30 degrees C.

Enzymatic Synthesis of Enantiopure α‐ and β‐Amino Acids by Phenylalanine Aminomutase‐Catalysed Amination of Cinnamic Acid Derivatives

“…To explain the retention of configuration at both reaction centres, Walker and co-workers proposed that the substrate might bind in the PAM active site with the carboxylate group and the phenyl ring arranged in a syn-periplanar orientation, which positions the migrating NH 3 and hydrogen groups on the same side of the substrate molecule. [28] This orientation could be consistent with (Z)-cinnamic acid as an intermediate. Based on this assumption, we expected (Z)-cinnamic acid to be a substrate for the ammonia addition reaction.…”

Mechanistic, Mutational, and Structural Evaluation of a Taxus Phenylalanine Aminomutase

“…8 In addition, the stereochemistry must account for the retention of configuration at the migration termini. 22 These data suggest that the amino group and hydrogen displaced from the (2S)-R-phenylalanine substrate must rebind, respectively, at C β and C R of the (E)-cinnamate before it diffuses from the active site. The constraints of the intramolecularity and stereochemistry of the TcPAM reaction present a challenge because the amino group must attach to the cinnamate on the side facing opposite to that of the NH 2 -MIO intermediate (cf.…”

“…The foregoing proposed mechanism is consistent with all of the stereochemical and mechanistic findings for TcPAM. In addition, the K eq ∼1 for the TcPAM reaction 22 suggests that the rotamers are energetically equivalent.…”

β‐Amino Acid Biosynthesis

“…So far, three types of 2,3-aminomutases are known [7,35]. The first type requires S-adenosylmethionine (SAM), pyridoxal 5 0 -phosphate (PLP), and iron-sulfur clusters as cofactors.…”