Engineering PQQ-glucose dehydrogenase into an allosteric electrochemical Ca²⁺ sensor

Abstract

Electrochemical biosensors convert biological events to an electrical current. To date most electrochemical biosensors exploit activities of naturally occurring enzymes. Here we demonstrated that insertion of a calmodulin domain into the redox enzyme PQQ-glucose dehydrogenase resulted in a selective Ca(2+) biosensor that could be used to rapidly measure Ca(2+) concentrations in human biological fluids. We were able to convert a point-of-care glucometer into Ca(2+) monitor by refurbishing it with the developed biosensor. We propose that similar engineering strategies may be used to create highly specific electrochemical biosensors to other analytes. Compatibility with cheap and ubiquitous amperometric detectors is expected to accelerate progression of these biosensors into clinical applications.

Bioelectrocatalytic Electrodes Modified with PQQ‐Glucose Dehydrogenase‐Calmodulin Chimera Switchable by Peptide Signals: Pathway to Generic Bioelectronic Systems Controlled by Biomolecular Inputs

“…Chimeric signal‐switchable enzymes with luminescent, fluorescent, proteolytic, and various other enzymatic outputs have been reported ,. This approach has been applied recently to construct pyrroloquinoline quinone‐dependent glucose dehydrogenase (PQQ‐GDH) (typical quinoprotein) fused with calmodulin (CaM) unit responsive to Ca 2+ cations ,. Since PQQ‐GDH enzyme is capable of direct (non‐mediated) electron transfer to various electrodes (particularly based on carbon nanotubes, “buckypaper”,, or on graphene‐functionalized electrodes), the chimeric PQQ‐GDH‐CaM enzyme was previously immobilized on a graphene‐modified carbon fiber electrode to yield the Ca 2+ ‐dependent electrochemical system .…”

Combinatorial design of chemical‐dependent protein switches for controlling intracellular electron transfer

“…Purified recombinant Fd switches that exhibit ligand‐induced shifts in midpoint potential can be coupled directly to materials to create miniature‐sensing devices. Engineered proteins have previously been used to build sensing devices by monitoring the ligand‐induced activity of glucose dehydrogenase using chronoamperometry . Additionally, Fd switches can be expressed in cells and used to regulate ET to partner proteins without the need for transcription and translation.…”

Mechanism of Reconstitution/Activation of the Soluble PQQ-Dependent Glucose Dehydrogenase from Acinetobacter calcoaceticus: A Comprehensive Study

“…10,20,21,22,23,24,25 Among the enzymes whose catalytic activity can be easily and efficiently switched on via reconstitution, the soluble quinoprotein glucose dehydrogenase (sGDH, code UniprotKB F0KFV3) is certainly the most prevailing and attractive. 15,26,27,28,29,30,31,32,33 The reason for such interest is that, in the presence of calcium ions, the catalytic property of sGDH for aldoses oxidation can be rapidly and spontaneously activated through the specific and tight binding of its PQQ cofactor to the apoprotein (apo-sGDH). This property has led to the design of novel analytical methods for the sensitive detection of calcium ions 30 or PQQ, 29 and to the conception of unique signal amplification strategies to boost the analytical performances of miscellaneous affinity binding assays.…”

“…15,26,27,28,29,30,31,32,33 The reason for such interest is that, in the presence of calcium ions, the catalytic property of sGDH for aldoses oxidation can be rapidly and spontaneously activated through the specific and tight binding of its PQQ cofactor to the apoprotein (apo-sGDH). This property has led to the design of novel analytical methods for the sensitive detection of calcium ions 30 or PQQ, 29 and to the conception of unique signal amplification strategies to boost the analytical performances of miscellaneous affinity binding assays. 15,31,32 Other features which makes sGDH an attractive activatable enzyme is the ease with which the apoenzyme can be overproduced in a recombinant strain of Escherichia coli and isolated with a high yield and purity (totally free of PQQ).…”

“…29 Also, the holoenzyme exhibits a remarkably high catalytic activity towards the oxidation of glucose with concomitant reduction of a wide range of natural or artificial electron acceptors, a catalytic reactivity that can be easily monitored either spectrophotometrically or electrochemically. Albeit there are several works reporting on sGDH reconstitution 34,36,37,38 as well as on its exploitation in different biotechnological applications, 15,29,30,31,32 its reconstitution mechanism remains unknown. Previous studies have yet established that, similarly to alcohol dehydrogenase, reconstitution of the homodimeric sGDH enzyme requires the binding of six calcium ions and two PQQ molecules.…”