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

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.

Genome sequences of Arthrobacter spp. that use a modified sulfoglycolytic Embden–Meyerhof–Parnas pathway

“…IolG proteins are NAD(P)-dependent oxidoreductases of the Gfo/Idh/MocA family, and catalyze oxidation of the hydroxyl groups of pyranose and inositol rings (Taberman et al 2016 ). Members of this family include inositol dehydrogenase (Idh), which forms 2-keto-myo-inositol (2-inosose) from myo -inositol (Ramaley et al 1979 ; Yoshida et al 2006 ), glucose-6-phosphate dehydrogenase (G6PD), which forms 6-phosphogluconolactone from glucose-6-phosphate (Rowland et al 1994 ), and levoglucosan dehydrogenase, which forms 3-keto-levoglucosan from levoglucosan (1,6-anhydro-β- d -glucose) (Sugiura et al 2018 ; Kuritani et al 2020 ). Inositol dehydrogenase IolG from Bacillus subtilis has maximal activity on myo -inositol and possesses activity on d -glucose and d -xylose, and produces d -gluconolactone from the former (Ramaley et al 1979 ).…”

Identification of levoglucosan degradation pathways in bacteria and sequence similarity network analysis

“…All LG-degrading bacteria studied at a genetic level contain close homologs of lgdA, and previous work has sought to identify genes involved in LG transport and metabolism by analysis of the surrounding genomic regions. In the case of Bacillus smithii S-270M, the gene cluster containing lgdA-lgdB1-lgdB2-lgdC also contains genes encoding ABC transporter subunits and an ABC transporter substrate binding protein (Kuritani et al 2020). However, other LG degrading organisms such as P. phenanthenivorans Sphe3, Paenibacillus athensensis MEC069, Shinella sumterensis MEC087, Microbacterium MEC084, and Klebsiella pneumoniae MEC097 contain lgdA-lgdB1 and genes encoding ABC transporter subunits and substrate binding proteins, but homologs of lgdB2 and lgdC were not identified (Arya et al 2022), raising questions as to whether other enzymes may be used for the final steps of LG metabolism.…”

Both levoglucosan kinase activity and transport capacity limit the utilization of levoglucosan in Saccharomyces cerevisiae

“…To date, two pathways for the metabolism of levoglucosan have been identified (Fig. 1 ) [ 10 , 11 ]. First, levoglucosan is phosphorylated into glucose-6-phosphate by levoglucosan kinase (LGK) or 1,6-anhydro-N-acetylmuramic acid kinase (AnmK).…”

“…Second, levoglucosan can be sequentially metabolized through oxidation, β-elimination, hydration, and reduction. However, the enzymes involved in this pathway are not well studied except for levoglucosan dehydrogenase (LGDH) [ 11 , 14 ]. Some proteins with LGK activity have been reported, including proteins from Rhodosporidium toruloides , Rhodotorula glutinis [ 15 ], Aspergillus terreus [ 16 ], Aspergillus niger [ 17 ], and Lipomyces starkeyi [ 18 ].…”

“…Second, levoglucosan can be sequentially metabolized through oxidation, β-elimination, hydration, and reduction. However, the enzymes involved in this pathway are not well studied except for levoglucosan dehydrogenase (LGDH) [11,14]. Some proteins with LGK…”