Purification and Characteristics of a NAD(P)-Dependent Gluconate 5-Dehydrogenase from Thermotoga maritima MSB8

Abstract

: NADP-dependent gluconate 5-dehydrogenase catalyzes the reversible oxidoreduction reaction between D-gluconate and 5-keto-D-gluconate. NADP-dependent gluconate 5-dehydrogenase from bacteria and fungi has been reported. The reported enzyme showed activity only for NADP as the cofactor. This study purified and characterized a NAD(P)-dependent gluconate 5-dehydrogenase (TmGNDH) from the archaea Thermotoga maritima MSB8. TmGNDH is composed of two identical subunits, and a single subunit has 255 amino acids with molecular mass of 28 kDa. TmGNDH is affiliated with the short-chain dehydrogenase/reductase family. Purified TmGNDH has a specific activity of 102.8 U/mg. The enzyme preferred NADP to NAD as its cofactor. TmGNDH exhibited a Km value of 121.3 ± 3.0 mM and 𝑘 𝑐𝑎𝑡 value of 3181 ± 34.1 𝑠 −1 toward D-gluconate as a substrate. TmGNDH had lower activity with D-sorbitol, D-xylitol, D-glucose and D-xylose as substrates. The optimal pH and temperature of TmGNDH were observed at pH 9.0 and 60℃ with NADP as the cofactor . The thermostability of the enzyme suggests that it can be used as an industrially effective tool for synthesizing 5-keto-D-gluconate. gluconate 5-dehydrogenase has not been reported. In this study, we purified and characterized a thermostable NAD(P)-dependent gluconate 5-dehydrogenase (TmGNDH) from the archaea Thermotoga maritima MSB8. The crystal structure (PDB ID: 1VL8) of the enzyme has been resolved, but its biochemical properties have not been reported.