Cβ-Selective Aldol Addition of D-Threonine Aldolase by Spatial Constraint of Aldehyde Binding

초록

D-Threonine aldolase (DTA) is a useful biocatalyst that reversibly converts glycine and aldehyde to beta-hydroxy-alpha-D-amino acid. However, low activity and poor diastereoselectivity limit its applications. Here we report DTA from Filomicrobium marinum (FmDTA) that shows much higher activity and C beta-stereoselectivity in D-threonine production compared with those of other known DTAs. We determine the FmDTA structure at a 2.2 angstrom resolution and propose a DTA catalytic mechanism with a kernel of the Lys49 inner proton sink and metal ion in the aldol reaction cycle. The enzyme is rationally engineered to have high C beta-stereoselectivity based on spatial constraint at the anti-specific aldehyde position in the mechanism, and the rational strategy is further applied to other DTAs for syn-production. The final FmDTA(G179A/S312A) variant exhibits a near-perfect 99.5% de value for D-threonine and maintains the de value above 93% even under kinetically unfavorable conditions. This study demonstrates how a detailed understanding of the reaction mechanism can be used for rational protein engineering.

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