Crystal structure of an activity-enhanced hyperthermophilic quinoprotein aldose sugar dehydrogenase
TL;DR
The crystal structures of R64Q/D350N in both the PQQ-unbound and bound forms were determined to elucidate the molecular mechanism underlying these enhancements and indicate that the increased activity and reduced activity of the R64Q/D350N mutant are likely attributable to the enhanced Ca 2+ uptake into the active site and a significant change in the position of R352.
Abstract
Pyrroloquinoline quinone (PQQ)-dependent aldose sugar dehydrogenase (PaeASD) from the hyperthermophilic archaeon Pyrobaculum aerophilum has great potential for durable bioelectronic devices because of its exceptionally high stability even at high temperatures and across a wide pH range. However, the PaeASD-immobilized electrode has less current output because of the enzyme's low catalytic activity that limits its practical application. A directed evolution approach produced a double mutant (R64Q/D350N) that exhibited a 2.4-fold higher maximum reaction velocity and 3.8-fold lower Michaelis constant ( K m ) for glucose than those of the original enzyme. In this study, we determined the crystal structures of R64Q/D350N in both the PQQ-unbound and bound forms to elucidate the molecular mechanism underlying these enhancements. The mutant enzyme differed notably from the wild-type, with a loop (N350 − R352) shifting away from the PQQ-binding site. The glucose-binding model indicates that, in the active site of the wild-type enzyme, the bulky side chain of R352 is positioned between the C2 carboxyl and C3 hydroxyl groups of PQQ and glucose, respectively, thereby restricting glucose binding. However, this barrier was not observed in the mutant enzyme. Additionally, Ca 2+ was retained at the active site of the mutant but not in the wild-type. Structural comparison and site-directed mutagenesis indicate that the increased activity and reduced K m of the R64Q/D350N mutant are likely attributable to the enhanced Ca 2+ uptake into the active site and a significant change in the position of R352, respectively. • Primary factors responsible for the enhanced activity observed in the R64Q/D350N mutant of PaeASD were analyzed by crystal structure analysis • Significant alterations in the R352 position are responsible for the reduced Km value • Increase in Ca2⁺ uptake into the active region contributes to the enhanced Vmax value