Enzymatic characteristics of d-mannose 2-epimerase, a new member of the acylglucosamine 2-epimerase superfamily
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Carbohydrate epimerases and isomerases are essential for the metabolism and synthesis of carbohydrates. In this study, Runella slithyformis Runsl_4512 and Dyadobacter fermentans Dfer_5652 were characterized from a cluster of uncharacterized proteins of the acylglucosamine 2-epimerase (AGE) superfamily. These proteins catalyzed the intramolecular conversion of d-mannose to d-glucose, whereas they did not act on β-(1 → 4)-mannobiose, N-acetyl-d-glucosamine, and d-fructose, which are substrates of known AGE superfamily members. The kcat/Km values of Runsl_4512 and Dfer_5652 for d-mannose epimerization were 3.89 and 3.51 min−1 mM−1, respectively. Monitoring the Runsl_4512 reaction through 1H-NMR showed the formation of β-d-glucose and β-d-mannose from d-mannose and d-glucose, respectively. In the reaction with β-d-glucose, β-d-mannose was produced at the initial stage of the reaction, but not in the reaction with α-d-glucose. These results indicate that Runsl_4512 catalyzed the 2-epimerization of the β-anomer substrate with a net retention of the anomeric configuration. Since 2H was obviously detected at the 2-C position of d-mannose and d-glucose in the equilibrated reaction mixture produced by Runsl_4512 in 2H2O, this enzyme abstracts 2-H from the substrate and adds another proton to the intermediate. This mechanism is in accordance with the mechanism proposed for the reactions of other epimerases of the AGE superfamily, that is, AGE and cellobiose 2-epimerase. Upon reaction with 500 g/L d-glucose at 50 °C and pH 8.0, Runsl_4512 and Dfer_5652 produced d-mannose with a 24.4 and 22.8% yield, respectively. These d-mannose yields are higher than those of other enzyme systems, and ME acts as an efficient biocatalyst for producing d-mannose.
Keywordsd-mannose Epimerase Cellobiose 2-epimerase Acylglucosamine 2-epimerase d-mannose isomerase
We thank Dr. Eri Fukushi of the GC-MS & NMR Laboratory, Research Faculty of Agriculture, Hokkaido University for NMR data analysis; Mr. Yusuke Takada of the DNA sequencing facility of the Research Faculty of Agriculture, Hokkaido University for assistance with DNA sequence analysis; and Ms. Nozomi Takeda of the Global Facility Center, Hokkaido University for the amino acid analysis. We would like to thank Editage (www.editage.jp) for English language editing.
This work was supported in part by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan [grant number 18K05382].
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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