Properties of recombinant Strep-tagged and untagged hyperthermophilic D-arabitol dehydrogenase from Thermotoga maritima.

ABSTRACT

The first hyperthermophilic D-arabitol dehydrogenase from Thermotoga maritima was heterologously purified from Escherichia coli. The protein was purified with and without a Strep-tag. The enzyme exclusively catalyzed the NAD(H)-dependent oxidoreduction of D-arabitol, D-xylitol, D-ribulose, or D-xylulose. A twofold increase of catalytic rates was observed upon addition of Mg(2+) or K(+). Interestingly, only the tag-less protein was thermostable, retaining 90% of its activity after 90 min at 85 °C. However, the tag-less form of D-arabitol dehydrogenase had similar kinetic parameters compared to the tagged enzyme, demonstrating that the Strep-tag was not deleterious to protein function but decreased protein stability. A single band at 27.6 kDa was observed on SDS-PAGE and native PAGE revealed that the protein formed a homohexamer and a homododecamer. The enzyme catalyzed oxidation of D-arabitol to D -ribulose and therefore belongs to the class of D-arabitol 2-dehydrogenases, which are typically observed in yeast and not bacteria. The product D-ribulose is a rare ketopentose sugar that has numerous industrially applications. Given its thermostability and specificity, D-arabitol 2-dehydrogenase is a desirable biocatalyst for the production of rare sugar precursors.