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

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

Research output: Contribution to journalArticleResearchpeer-review

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.

Original languageEnglish
Pages (from-to)596-603
Number of pages8
JournalChemBioChem
Volume19
Issue number6
DOIs
Publication statusPublished - 16 Mar 2018

Keywords

  • carbohydrates
  • conformation analysis
  • enzyme catalysis
  • kinases
  • reaction mechanisms

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Organic Chemistry

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