TM0416, a hyperthermophilic promiscuous nonphosphorylated sugar isomerase, catalyzes various C5 and C6 epimerization reactions

Sun Mi Shin, Thinh Phat Cao, Jin Myung Choi, Seong Bo Kim, Sang Jae Lee, Sung Haeng Lee, Dong Woo Lee

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9 Citations (Scopus)

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.

Original languageEnglish
Article numbere03291-16
Number of pages16
JournalApplied and Environmental Microbiology
Volume83
Issue number10
DOIs
Publication statusPublished - 1 May 2017
Externally publishedYes

Keywords

  • D-erythrose isomerase
  • D-tagatose 3-epimerase
  • L-ribulose 3-epimerase
  • Low-calorie sweeteners
  • Rare sugars
  • Thermotoga maritima

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