Mechanism of reconstitution/activation of the soluble PQQ-dependent glucose dehydrogenase from Acinetobacter calcoaceticus: a comprehensive study

TL;DR

This study uncovers the detailed mechanism of enzyme reconstitution of soluble PQQ-dependent glucose dehydrogenase from Acinetobacter calcoaceticus, revealing how cofactors bind and activate the enzyme through kinetic and structural analysis.

Contribution

It provides the first comprehensive kinetic and structural insights into the reconstitution mechanism of apo-sGDH with its cofactors PQQ and Ca2+.

Findings

Reconstitution exhibits biphasic kinetics with two activation pathways.

PQQ binding impedes Ca2+ access, slowing reconstitution.

Mutating P248 accelerates calcium insertion and enzyme activation.

Abstract

The ability to switch on the activity of an enzyme through its spontaneous reconstitution has proven to be a valuable tool in fundamental studies of enzyme structure/reactivity relationships or in the design of artificial signal transduction systems in bioelectronics, synthetic biology, or bioanalytical applications. In particular, those based on the spontaneous reconstitution/activation of the apo-PQQ-dependent soluble glucose dehydrogenase (sGDH) from Acinetobacter calcoaceticus were widely developed. However, the reconstitution mechanism of sGDH with its two cofactors, i.e. the pyrroloquinoline quinone (PQQ) and Ca2+, remains unknown. The objective here is to elucidate this mechanism by stopped-flow kinetics under single-turnover conditions. The reconstitution of sGDH exhibited biphasic kinetics, characteristic of a square reaction scheme associated to two activation pathways. From a complete kinetic analysis, we were able to fully predict the reconstitution dynamic, but also to demonstrate that when PQQ first binds to the apo-sGDH, it strongly impedes the access of Ca2+ to its enclosed position at the bottom of the enzyme binding site, thereby greatly slowing down the reconstitution rate of sGDH. This slow calcium insertion may purposely be accelerated by providing more flexibility to the Ca2+ binding loop through the specific mutation of the calcium coordinating P248 proline residue, reducing thus the kinetic barrier to calcium ion insertion. The dynamic nature of the reconstitution process is also supported by the observation of a clear loop shift and a reorganization of the hydrogen bonding network and van der Waals interactions observed in both active sites of the apo and holo forms, a structural change modulation that was revealed from the refined X-ray structure of apo-sGDH (PDB:5MIN).