Producing Glucose 6-phosphate from cellulosic biomass: Structural insights into levoglucosan bioconversion

John Paul Bacik, Justin R. Klesmith, Timothy A. Whitehead, Laura R. Jarboe, Clifford J. Unkefer, Brian L. Mark, Ryszard Michalczyk

    Research output: Research - peer-reviewArticle

    • 3 Citations

    Abstract

    The most abundant carbohydrate product of cellulosic biomass pyrolysis is the anhydrosugar levoglucosan (1, 6-anhydro-β-D-glucopyranose), which can be converted to glucose 6-phosphate by levoglucosan kinase (LGK). In addition to the canonical kinase phosphotransfer reaction, the conversion requires cleavage of the 1, 6-anhydro ring to allow ATP-dependent phosphorylation of the sugar O6 atom. Using x-ray crystallography, we show that LGK binds two magnesium ions in the active site that are additionally coordinated with the nucleotide and water molecules to result in ideal octahedral coordination. To further verify the metal binding sites, we co-crystallized LGK in the presence of manganese instead of magnesium and solved the structure de novo using the anomalous signal from four manganese atoms in the dimeric structure. The first metal is required for catalysis, whereas our work suggests that the second is either required or significantly promotes the catalytic rate. Although the enzyme binds its sugar substrate in a similar orientation to the structurally related 1, 6-anhydro-N-acetylmuramic acid kinase (AnmK), it forms markedly fewer bonding interactions with the substrate. In this orientation, the sugar is in an optimal position to couple phosphorylation with ring cleavage. We also observed a second alternate binding orientation for levoglucosan, and in these structures, ADP was found to bind with lower affinity. These combined observations provide an explanation for the high Km of LGK for levoglucosan. Greater knowledge of the factors that contribute to the catalytic efficiency of LGK can be used to improve applications of this enzyme for levoglucosan-derived biofuel production.

    LanguageEnglish (US)
    Pages26638-26648
    Number of pages11
    JournalJournal of Biological Chemistry
    Volume290
    Issue number44
    DOIs
    StatePublished - Oct 30 2015

    Profile

    Bioconversion
    Glucose-6-Phosphate
    Biomass
    1,6-anhydro-beta-glucopyranose
    Phosphotransferases
    Sugars
    Phosphorylation
    Manganese
    Magnesium
    Metals
    Atoms
    Substrates
    Enzymes
    Crystallography
    Biofuels
    Adenosine Diphosphate
    Catalysis
    Pyrolysis
    Nucleotides
    Adenosine Triphosphate

    ASJC Scopus subject areas

    • Biochemistry
    • Cell Biology
    • Molecular Biology

    Cite this

    Bacik, J. P., Klesmith, J. R., Whitehead, T. A., Jarboe, L. R., Unkefer, C. J., Mark, B. L., & Michalczyk, R. (2015). Producing Glucose 6-phosphate from cellulosic biomass: Structural insights into levoglucosan bioconversion. Journal of Biological Chemistry, 290(44), 26638-26648. DOI: 10.1074/jbc.M115.674614

    Producing Glucose 6-phosphate from cellulosic biomass : Structural insights into levoglucosan bioconversion. / Bacik, John Paul; Klesmith, Justin R.; Whitehead, Timothy A.; Jarboe, Laura R.; Unkefer, Clifford J.; Mark, Brian L.; Michalczyk, Ryszard.

    In: Journal of Biological Chemistry, Vol. 290, No. 44, 30.10.2015, p. 26638-26648.

    Research output: Research - peer-reviewArticle

    Bacik, JP, Klesmith, JR, Whitehead, TA, Jarboe, LR, Unkefer, CJ, Mark, BL & Michalczyk, R 2015, 'Producing Glucose 6-phosphate from cellulosic biomass: Structural insights into levoglucosan bioconversion' Journal of Biological Chemistry, vol 290, no. 44, pp. 26638-26648. DOI: 10.1074/jbc.M115.674614
    Bacik JP, Klesmith JR, Whitehead TA, Jarboe LR, Unkefer CJ, Mark BL et al. Producing Glucose 6-phosphate from cellulosic biomass: Structural insights into levoglucosan bioconversion. Journal of Biological Chemistry. 2015 Oct 30;290(44):26638-26648. Available from, DOI: 10.1074/jbc.M115.674614
    Bacik, John Paul ; Klesmith, Justin R. ; Whitehead, Timothy A. ; Jarboe, Laura R. ; Unkefer, Clifford J. ; Mark, Brian L. ; Michalczyk, Ryszard. / Producing Glucose 6-phosphate from cellulosic biomass : Structural insights into levoglucosan bioconversion. In: Journal of Biological Chemistry. 2015 ; Vol. 290, No. 44. pp. 26638-26648
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    abstract = "The most abundant carbohydrate product of cellulosic biomass pyrolysis is the anhydrosugar levoglucosan (1, 6-anhydro-β-D-glucopyranose), which can be converted to glucose 6-phosphate by levoglucosan kinase (LGK). In addition to the canonical kinase phosphotransfer reaction, the conversion requires cleavage of the 1, 6-anhydro ring to allow ATP-dependent phosphorylation of the sugar O6 atom. Using x-ray crystallography, we show that LGK binds two magnesium ions in the active site that are additionally coordinated with the nucleotide and water molecules to result in ideal octahedral coordination. To further verify the metal binding sites, we co-crystallized LGK in the presence of manganese instead of magnesium and solved the structure de novo using the anomalous signal from four manganese atoms in the dimeric structure. The first metal is required for catalysis, whereas our work suggests that the second is either required or significantly promotes the catalytic rate. Although the enzyme binds its sugar substrate in a similar orientation to the structurally related 1, 6-anhydro-N-acetylmuramic acid kinase (AnmK), it forms markedly fewer bonding interactions with the substrate. In this orientation, the sugar is in an optimal position to couple phosphorylation with ring cleavage. We also observed a second alternate binding orientation for levoglucosan, and in these structures, ADP was found to bind with lower affinity. These combined observations provide an explanation for the high Km of LGK for levoglucosan. Greater knowledge of the factors that contribute to the catalytic efficiency of LGK can be used to improve applications of this enzyme for levoglucosan-derived biofuel production.",
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