2. Academic Validation
  3. Conversion of levoglucosan into glucose by the coordination of four enzymes through oxidation, elimination, hydration, and reduction

Conversion of levoglucosan into glucose by the coordination of four enzymes through oxidation, elimination, hydration, and reduction

  • Sci Rep. 2020 Nov 18;10(1):20066. doi: 10.1038/s41598-020-77133-8.
Yuya Kuritani 1,  Kohei Sato 1,  Hideo Dohra 2 3,  Seiichiro Umemura 4,  Motomitsu Kitaoka 5,  Shinya Fushinobu 6 7,  Nobuyuki Yoshida 8
Affiliations

Affiliations

  • 1 Department of Engineering, Graduate School of Integrated Science and Technology, Shizuoka University, 3-5-1 Johoku, Naka-ku, Hamamatsu, 432-8561, Japan.
  • 2 Department of Science, Graduate School of Integrated Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan.
  • 3 Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan.
  • 4 Nihon Shokuhin Kako Co., Ltd., 30 Tajima, Fuji, 417-8530, Japan.
  • 5 Faculty of Agriculture, Niigata University, 8050 Ikarashi 2-no-cho, Niigata, 950-2181, Japan.
  • 6 Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
  • 7 Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
  • 8 Department of Engineering, Graduate School of Integrated Science and Technology, Shizuoka University, 3-5-1 Johoku, Naka-ku, Hamamatsu, 432-8561, Japan. [email protected].
PMID: 33208778 DOI: 10.1038/s41598-020-77133-8
Abstract

Levoglucosan (LG) is an anhydrosugar produced through glucan pyrolysis and is widely found in nature. We previously isolated an LG-utilizing thermophile, Bacillus smithii S-2701M, and suggested that this bacterium may have a metabolic pathway from LG to glucose, initiated by LG dehydrogenase (LGDH). Here, we completely elucidated the metabolic pathway of LG involving three novel enzymes in addition to LGDH. In the S-2701M genome, three genes expected to be involved in the LG metabolism were found in the vicinity of the LGDH gene locus. These four genes including LGDH gene (lgdA, lgdB1, lgdB2, and lgdC) were expressed in Escherichia coli and purified to obtain functional recombinant proteins. Thin layer chromatography analyses of the reactions with the combination of the four enzymes elucidated the following metabolic pathway: LgdA (LGDH) catalyzes 3-dehydrogenation of LG to produce 3-keto-LG, which undergoes β-elimination of 3-keto-LG by LgdB1, followed by hydration to produce 3-keto-D-glucose by LgdB2; next, LgdC reduces 3-keto-D-glucose to glucose. This sequential reaction mechanism resembles that proposed for an Enzyme belonging to glycoside hydrolase family 4, and results in the observational hydrolysis of LG into glucose with coordination of the four enzymes.

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