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

• Autores: Sun-Mi Shin, Thinh-Phat Cao, Jin Myung Choi, Giuseppe Seong-Bo Kim, Giuseppe Sang-Jae Lee, Sung Haeng Lee, Dong-Woo Lee
• Localización: Applied and Environmental Microbiology, ISSN 0099-2240, Vol. 83, Nº 10, 2017
• Idioma: inglés
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• Resumen

  • 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.