2-keto-3-L-arabinonate dehydratase (L-KdpD) and 2-keto-3-D-xylonate dehydratase (D-KdpD) are the third enzymes in the Weimberg pathway catalyzing the dehydration of respective 2-keto-3-deoxy sugar acids (KDP) to alpha-ketoglutaric semialdehyde (KGSA). The Weimberg pathway has been explored recently with respect to the synthesis of chemicals from L-arabinose and D-xylose. However, only limited work has been done toward characterizing these two enzymes. In this work, several new L-KdpDs and D-KdpDs were cloned and heterologously expressed inEscherichia coli. Following kinetic characterizations and kinetic stability studies, the L-KdpD fromCupriavidus necator(CnL-KdpD) and D-KdpD fromPseudomonas putida(PpD-KdpD) appeared to be the most promising variants from each enzyme class. Magnesium had no effect onCnL-KdpD, whereas increased activity and stability were observed forPpD-KdpD in the presence of Mg2+. Furthermore,CnL-KdpD was not inhibited in the presence of L-arabinose and L-arabinonate, whereasPpD-KdpD was inhibited with D-xylonate (I(50)of 75 mM), but not with D-xylose. Both enzymes were shown to be highly active in the one-step conversions of L-KDP and D-KDP.CnL-KdpD converted > 95% of 500 mM L-KDP to KGSA in the first 2 h whilePpD-KdpD converted > 90% of 500 mM D-KDP after 4 h. Both enzymes in combination were able to convert 83% of a racemic mixture of D,L-KDP (500 mM) after 4 h, with both enzymes being specific toward the respective stereoisomer.