Characterization of highly active 2-keto-3-deoxy-L-arabinonate and 2-keto-3-deoxy-D-xylonate dehydratases in terms of the biotransformation of hemicellulose sugars to chemicals

Abstract

Abstract 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 α-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 in Escherichia coli . Following kinetic characterizations and kinetic stability studies, the L-KdpD from Cupriavidus necator ( Cn L-KdpD) and D-KdpD from Pseudomonas putida ( Pp D-KdpD) appeared to be the most promising variants from each enzyme class. Magnesium had no effect on Cn L-KdpD, whereas increased activity and stability were observed for Pp D-KdpD in the presence of Mg 2+ . Furthermore, Cn L-KdpD was not inhibited in the presence of L-arabinose and L-arabinonate, whereas Pp D-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. Cn L-KdpD converted > 95% of 500 mM L-KDP to KGSA in the first 2 h while Pp D-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. Key points • L-KdpDs and D-KdpDs are specific toward L- and D-KDP, respectively. • Mg 2+ affected activity and stabilities of D-KdpDs, but not of L-KdpDs. • CnL-KdpD and PpD-KdpD converted 0.5 M of each KDP isomer reaching 95 and 90% yield. • Both enzymes in combination converted 0.5 M racemic D,L-KDP reaching 83% yield.

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