Reconstitution of Membrane-Integrated Quinoprotein Glucose Dehydrogenase Apoenzyme with PQQ and the Holoenzyme's Mechanism of Action

Abstract

Membrane-integrated quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus was produced by heterologous expression of the gene for it in an Escherichia coli recombinant strain. The apoenzyme (lacking the cofactor pyrroloquinoline quinone, PQQ) was solubilized with Triton X-100 and purified to homogeneity. Reconstitution of the apoenzyme to full activity in the assay was achieved with a stoichiometric amount of PQQ in the presence of Mg2+. Just as for other PQQ-containing dehydrogenases where Ca2+ fulfills this role, Mg2+ anchors PQQ to the mGDH protein and activates the bound cofactor. This occurs in a precise way since high anomer specificity was found for the enzyme toward the sugars tested. Although the steady-state-type kinetics were as expected for a dye-linked dehydrogenase (ping-pong) and the PQQ in it was present in oxidized form, addition of glucose to the holoenzyme resulted in a very slow but continuous production of gluconolactone; i.e., the reaction did not stop after one turnover, with O2 apparently acting as an (albeit poor) electron acceptor by reoxidizing PQQH2 in the enzyme. The surprisingly low reactivity with glucose, in the absence of dye, as compared to the activity observed in the steady-state assay appeared to be due to formation of an anomalous enzyme form, mGDH. Formation of normal holoenzyme, mGDH, reducing added glucose immediately to gluconolactone (in one turnover), was achieved by treating mGDH with sulfite, by reconstituting apoenzyme with PQQ in the presence of sulfite, or by applying assay conditions to mGDH (addition of PMS/DCPIP). As compared to other quinoprotein dehydrogenases, mGDH appears to be unique with respect to the mode of PQQ-binding, as expressed by the special conditions for reconstitution and the absorption spectra of the bound cofactor, and the reactivity of the reduced enzyme toward O2. The primary cause for this seems not to be related to a different preference for the activating bivalent metal ion but to the special way of binding of PQQ to mGDH.

Regulation of Pyrroloquinoline Quinone-Dependent Glucose Dehydrogenase Activity in the Model Rhizosphere-Dwelling Bacterium Pseudomonas putida KT2440

“…Purified GDH enzymes from other Gram-negative bacteria (including E. coli, Enterobacter asburiae, Erwinia sp. 34-1, Acinetobacter calcoaceticus) gave K m values with glucose in the range of 1.1 mM to 4.0 mM (32,33). While less information is available about K m values with PQQ for quinoproteins in general, the measured value of 1.1 M for PQQ with GDH from E. coli DH5␣ falls within the range of values measured with other E. coli strains, including gcd mutants, at 0.05 to 21 M (34-36), and is in (Tables 2 and 3) are sufficient to saturate the enzyme under steady-state conditions.…”

Kinetic Analysis of Semisynthetic Peroxidase Enzymes Containing a Covalent DNA–Heme Adduct as the Cofactor

“…[16] Owing to the role of prosthetic groups in the catalytic activity of many enzymes, intensive research has been carried out within the last 50 years to investigate how structural alterations influence the properties of the respective enzymes. Examples include flavoenzymes, [17,18] pyrroloquinoline quinine, [19,20] and heme-containing [21,22] enzymes. Heme enzymes are particularly interesting as they catalyze a variety of redox reactions and are therefore powerful catalysts for synthetic organic chemistry.…”

Boolean Logic Networks Mimicked with Chimeric Enzymes Activated/Inhibited by Several Input Signals

“…The enzyme assay was performed according to the standard procedure . The optical absorbance was measured at λ=600 nm corresponding to the maximum absorbance of DCPIP.…”