TY - JOUR
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
Y1 - 2018/3/16
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
UR - http://www.scopus.com/inward/record.url?scp=85041055171&partnerID=8YFLogxK
U2 - 10.1002/cbic.201700587
DO - 10.1002/cbic.201700587
M3 - Article
AN - SCOPUS:85041055171
SN - 1439-4227
VL - 19
SP - 596
EP - 603
JO - ChemBioChem
JF - ChemBioChem
IS - 6
ER -