Structural Insights into the Mechanism of Tartrate Dehydrogenase: A versatile enzyme catalyzing multiple reactions

Abstract

Tartrate Dehydrogenase (TDH) is a member of the family of R‐hydroxy acid dehydrogenases, occupying a unique branch. TDH catalyzes several interesting transformations which vary depending on the nature and the stereochemistry of the respective substrates. Structural and functional information on this unusual enzyme will provide a better understanding of the evolution of enzymes within this species, as well as guide its engineering for chiral synthon production. Here we report the first structure of TDH solved to 2.0 Åresolution by SAD phasing using selenomethionine labeled protein. The enzyme is a dimer with residues from both monomers forming each active site cleft. The complex with NADH, Mg2+ and the intermediate analog oxalate represents the closed state of the enzyme. Based on the structures we have identified putative substrate and metal binding residues. Their proposed roles in the catalytic mechanism have been confirmed by site directed mutagenesis and kinetic analysis. In particular, the role of D225 as the base responsible for initial proton abstraction from the C2 hydroxyl of the substrate has now been established. A product complex provides additional insights which complement our mechanistic picture of this unusual enzyme. These results will now be used to guide the reengineering of this enzyme.