Biochemical Characterization and Mechanistic Analysis of the Levoglucosan Kinase from Lipomyces starkeyi

Christina Rother, Alexander Gutmann, Ramakrishna Gudiminchi, Hansjörg Weber, Alexander Lepak, Bernd Nidetzky

Publikation: Beitrag in einer FachzeitschriftArtikelForschungBegutachtung

Abstract

Levoglucosan kinase (LGK) catalyzes the simultaneous hydrolysis and phosphorylation of levoglucosan (1,6-anhydro-β-d-glucopyranose) in the presence of Mg2+–ATP. For the Lipomyces starkeyi LGK, we show here with real-time in situ NMR spectroscopy at 10 °C and pH 7.0 that the enzymatic reaction proceeds with inversion of anomeric stereochemistry, resulting in the formation of α-d-glucose-6-phosphate in a manner reminiscent of an inverting β-glycoside hydrolase. Kinetic characterization revealed the Mg2+ concentration for optimum activity (20–50 mm), the apparent binding of levoglucosan (Km=180 mm) and ATP (Km=1.0 mm), as well as the inhibition by ADP (Ki=0.45 mm) and d-glucose-6-phosphate (IC50=56 mm). The enzyme was highly specific for levoglucosan and exhibited weak ATPase activity in the absence of substrate. The equilibrium conversion of levoglucosan and ATP lay far on the product side, and no enzymatic back reaction from d-glucose-6-phosphate and ADP was observed under a broad range of conditions. 6-Phospho-α-d-glucopyranosyl fluoride and 6-phospho-1,5-anhydro-2-deoxy-d-arabino-hex-1-enitol (6-phospho-d-glucal) were synthesized as probes for the enzymatic mechanism but proved inactive with the enzyme in the presence of ADP. The pyranose ring flip 4C11C4 required for 1,6-anhydro-product synthesis from d-glucose-6-phosphate probably presents a major thermodynamic restriction to the back reaction of the enzyme.

Originalspracheenglisch
Seiten (von - bis)596-603
Seitenumfang8
FachzeitschriftChemBioChem
Jahrgang19
Ausgabenummer6
DOIs
PublikationsstatusVeröffentlicht - 16 Mär 2018

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Lipomyces
Glucose-6-Phosphate
Phosphotransferases
Adenosine Diphosphate
Adenosine Triphosphate
Enzymes
Stereochemistry
Phosphorylation
Glycoside Hydrolases
Fluorides
Thermodynamics
Nuclear magnetic resonance spectroscopy
Inhibitory Concentration 50
Adenosine Triphosphatases
1,6-anhydro-beta-glucopyranose
Hydrolysis
Magnetic Resonance Spectroscopy
Kinetics
Substrates

Schlagwörter

    ASJC Scopus subject areas

    • !!Biochemistry
    • !!Molecular Medicine
    • !!Molecular Biology
    • Organische Chemie

    Dies zitieren

    Biochemical Characterization and Mechanistic Analysis of the Levoglucosan Kinase from Lipomyces starkeyi. / Rother, Christina; Gutmann, Alexander; Gudiminchi, Ramakrishna; Weber, Hansjörg; Lepak, Alexander; Nidetzky, Bernd.

    in: ChemBioChem, Jahrgang 19, Nr. 6, 16.03.2018, S. 596-603.

    Publikation: Beitrag in einer FachzeitschriftArtikelForschungBegutachtung

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    T1 - Biochemical Characterization and Mechanistic Analysis of the Levoglucosan Kinase from Lipomyces starkeyi

    AU - Rother, Christina

    AU - Gutmann, Alexander

    AU - Gudiminchi, Ramakrishna

    AU - Weber, Hansjörg

    AU - Lepak, Alexander

    AU - Nidetzky, Bernd

    PY - 2018/3/16

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    N2 - Levoglucosan kinase (LGK) catalyzes the simultaneous hydrolysis and phosphorylation of levoglucosan (1,6-anhydro-β-d-glucopyranose) in the presence of Mg2+–ATP. For the Lipomyces starkeyi LGK, we show here with real-time in situ NMR spectroscopy at 10 °C and pH 7.0 that the enzymatic reaction proceeds with inversion of anomeric stereochemistry, resulting in the formation of α-d-glucose-6-phosphate in a manner reminiscent of an inverting β-glycoside hydrolase. Kinetic characterization revealed the Mg2+ concentration for optimum activity (20–50 mm), the apparent binding of levoglucosan (Km=180 mm) and ATP (Km=1.0 mm), as well as the inhibition by ADP (Ki=0.45 mm) and d-glucose-6-phosphate (IC50=56 mm). The enzyme was highly specific for levoglucosan and exhibited weak ATPase activity in the absence of substrate. The equilibrium conversion of levoglucosan and ATP lay far on the product side, and no enzymatic back reaction from d-glucose-6-phosphate and ADP was observed under a broad range of conditions. 6-Phospho-α-d-glucopyranosyl fluoride and 6-phospho-1,5-anhydro-2-deoxy-d-arabino-hex-1-enitol (6-phospho-d-glucal) were synthesized as probes for the enzymatic mechanism but proved inactive with the enzyme in the presence of ADP. The pyranose ring flip 4C1→1C4 required for 1,6-anhydro-product synthesis from d-glucose-6-phosphate probably presents a major thermodynamic restriction to the back reaction of the enzyme.

    AB - Levoglucosan kinase (LGK) catalyzes the simultaneous hydrolysis and phosphorylation of levoglucosan (1,6-anhydro-β-d-glucopyranose) in the presence of Mg2+–ATP. For the Lipomyces starkeyi LGK, we show here with real-time in situ NMR spectroscopy at 10 °C and pH 7.0 that the enzymatic reaction proceeds with inversion of anomeric stereochemistry, resulting in the formation of α-d-glucose-6-phosphate in a manner reminiscent of an inverting β-glycoside hydrolase. Kinetic characterization revealed the Mg2+ concentration for optimum activity (20–50 mm), the apparent binding of levoglucosan (Km=180 mm) and ATP (Km=1.0 mm), as well as the inhibition by ADP (Ki=0.45 mm) and d-glucose-6-phosphate (IC50=56 mm). The enzyme was highly specific for levoglucosan and exhibited weak ATPase activity in the absence of substrate. The equilibrium conversion of levoglucosan and ATP lay far on the product side, and no enzymatic back reaction from d-glucose-6-phosphate and ADP was observed under a broad range of conditions. 6-Phospho-α-d-glucopyranosyl fluoride and 6-phospho-1,5-anhydro-2-deoxy-d-arabino-hex-1-enitol (6-phospho-d-glucal) were synthesized as probes for the enzymatic mechanism but proved inactive with the enzyme in the presence of ADP. The pyranose ring flip 4C1→1C4 required for 1,6-anhydro-product synthesis from d-glucose-6-phosphate probably presents a major thermodynamic restriction to the back reaction of the enzyme.

    KW - carbohydrates

    KW - conformation analysis

    KW - enzyme catalysis

    KW - kinases

    KW - reaction mechanisms

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    U2 - 10.1002/cbic.201700587

    DO - 10.1002/cbic.201700587

    M3 - Article

    VL - 19

    SP - 596

    EP - 603

    JO - ChemBioChem

    JF - ChemBioChem

    SN - 1439-4227

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