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

• Published in: Applied and environmental microbiology Vol. 83; no. 10; p. E03291
• Main Authors: Shin, Sun-Mi, Cao, Thinh-Phat, Choi, Jin Myung, Kim, Seong-Bo, Lee, Sang-Jae, Lee, Sung Haeng, Lee, Dong-Woo
• Format: Journal Article
• Language: English
• Published: Washington American Society for Microbiology 01.05.2017
• Subjects: Accessibility; Alignment; Amino acid sequence; Amino acids; Binding; Biochemistry; Catalysis; Chemical reactions; Coding; Conversion; Coordination compounds; Crystal structure; D-tagatose; E coli; Enzymes; Enzymology and Protein Engineering; Epimerase; Escherichia coli; Genetics; Hydrophobicity; Inspection; Isomerization; L-ribulose; Manganese; Metal ions; Metals; Nucleotide sequence; Residues; Searching; Similarity; Sorbose; Substrate preferences; Substrate specificity; Sugar; Thermotoga maritima; Xylulose; Yield
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.03291-16

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 A. 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.