5h1w
Crystal Structure of Hyperthermophilic Thermotoga maritima L-Ketose-3-Epimerase with Mn2+ and L(+)-Erythrulose
Structural highlights
FunctionIOLO_THEMA Catalyzes the reversible epimerization between 5-keto-L-gluconate (5-dehydro-L-gluconate) and D-tagaturonate, and thus probably functions in a myo-inositol degradation pathway together with IolG, IolM and IolN (PubMed:23441918). Is not active on the enantiomer 5-keto-D-gluconate (PubMed:23441918). Was also shown to be a nonphosphorylated sugar isomerase with broad substrate specificity in vitro (PubMed:28258150). Is able to catalyze the reversible C3-epimerization of L-ribulose to L-xylulose, D-ribulose to D-xylulose, D-psicose to D-fructose, and D-tagatose to D-sorbose, with a substrate preference for ketopentoses rather than ketohexoses (PubMed:28258150). Also catalyzes the aldose-ketose isomerization reaction from either D-erythrose or D-threose to D-erythrulose (PubMed:28258150). Exhibits no activity for C4-epimerization of D-tagatose to D-fructose (PubMed:28258150).[1] [2] Publication Abstract from PubMedThere is currently little information on non-phosphorylated sugar epimerases, which are of potential interest for producing rare sugars. Herein, we found a gene (TM0416) encoding a putative d-tagatose 3-epimerase-related protein from a 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 non-phosphorylated sugar epimerase. The recombinant enzyme exhibited maximal 3-epimerization of l-ribulose to l-xylulose at around 80 degrees C and pH 7 in the presence of 1 mM Mn2+ In addition, this enzyme showed unusually high activity of the epimerization of d-tagatose to d-sorbose with a conversion yield of 20% after 6 h at 80 degrees 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 degrees C and 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 1.5 and 1.6 A resolutions. Detailed inspection of the structural features and biochemical data clearly demonstrated that this metalloenzyme with freely accessible substrate-binding site and the neighboring hydrophobic residues exhibits differential and promiscuous substrate preference when compared with its mesophilic counterparts. Therefore, this study suggests that TM0416 can be functionally classified as a novel type of l-ribulose 3-epimerase with d-erythrose isomerase activity.IMPORTANCE Rare sugars, which occur naturally in small amounts, have attracted considerable interest in the food and drug industries. However, there is little information on non-phosphorylated sugar epimerases, which might be potentially applied for the production of rare sugars. This study describes a characterization of a functional annotation of a putative non-phosphorylated sugar 3-epimerase from a hyperthermophilic bacterium. Furthermore, we determined its first crystal structures in complex with divalent metal ions and l-erythrulose, highlighting its metal-dependent bifunctional sugar-isomerizing activity. Consequently, this hyperthermophilic l-ribulose 3-epimerase (R3E) exhibited d-erythrose/d-threose isomerase activity, with structural features near the substrate-binding site distinct from its mesophilic counterparts. Moreover, this metalloenzyme showed unusually high activity for the epimerization of d-tagatose to d-sorbose at 70 degrees C. In this regard, TM0416 can be functionally classified as a novel type of a promiscuous R3E with a potential for the production of rare sugars in the food and pharmaceutical industries. TM0416 Is a Hyperthermophilic Promiscuous Non-phosphorylated Sugar Isomerase That Catalyzes Various C5 and C6 Epimerization Reactions.,Shin SM, Cao TP, Choi JM, Kim SB, Lee SJ, Lee SH, Lee DW Appl Environ Microbiol. 2017 Mar 3. pii: AEM.03291-16. doi: 10.1128/AEM.03291-16. PMID:28258150[3] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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