TM0416, a Hyperthermophilic Promiscuous Nonphosphorylated Sugar Isomerase, Catalyzes Various C5 and C6 Epimerization Reactions

Abstract
There is currently little information on nonphosphorylated sugar epimerases, which are of potential interest for producing rare sugars. We found a gene (the TM0416 gene) encoding a putative D-tagatose-3-epimerase-related protein from the hyperthermophilic bacterium Thermotoga maritima. We overexpressed the TM0416 gene in Escherichia coli and purified the resulting recombinant protein for detailed characterization. Amino acid sequence alignment and a structural similarity search revealed that TM0416 is a putative nonphosphorylated sugar epimerase. The recombinant enzyme exhibited maximal C-3 epimerization of L-ribulose to L-xylulose at 80°C and pH 7 in the presence of 1 mM Mn2. In addition, this enzyme showed unusually high activity for the epimerization of D-tagatose to D-sorbose, with a conversion yield of 20% after 6 h at 80°C. Remarkably, the enzyme catalyzed the isomerization of D-erythrose or D-threose to D-erythrulose significantly, with conversion yields of 71% and 54.5%, respectively, after 6 h at 80°C at pH 7. To further investigate the substrate specificity of TM0416, we determined its crystal structures in complex with divalent metal ions and L-erythrulose at resolutions of 1.5 and 1.6 Å. Detailed inspection of the structural features and biochemical data clearly demonstrated that this metalloenzyme, with a freely accessible substrate-binding site and neighboring hydrophobic residues, exhibits different and promiscuous substrate preferences, compared with its mesophilic counterparts. Therefore, this study suggests that TM0416 can be functionally classified as a novel type of L-ribulose 3-epimerase (R3E) with D-erythrose isomerase activity.