Production, characterization, and reconstitution of recombinant quinoprotein glucose dehydrogenase (soluble type; EC 1.1.99.17) apoenzyme of Acinetobacter calcoaceticus.
Soluble, periplasmic quinoprotein glucose dehydrogenase of Acinetobacter calcoaceticus (sGDH; EC 1.1.99.17) was produced in good yield in the apoenzyme form (without the cofactor pyrroloquinoline quinone, PQQ) by an Escherichia coli recombinant strain provided with a plasmid containing the gene under control of a lac promoter. Structural analysis of the purified apoenzyme revealed that the E. coli strain used produces the correct mature protein. Titration of the apoenzyme with PQQ in the presence of Ca2+ showed that a linear relation exists between the amount of added PQQ and activity observed, and that the subunit and PQQ associate in a molar ratio of 1:1. Based on spectral and enzymatic criteria, it is concluded that the present holoenzyme preparation has a better quality than the previously described preparations of authentic holoenzyme. As isolated here, the recombinant apoenzyme was in the dimeric form. Partial monomerization occurred upon gel filtration in a buffer with chelator and the process could be reversed with Ca2+. PQQ binds to the dimer in the presence of chelator, not to the monomer. However, the PQQ-containing dimer was not active and showed an unusual absorption spectrum which was slowly converted into a PQQH2-like spectrum when glucose was added. Full restoration of activity was achieved upon addition of Ca2+ and the spectra were immediately converted into those of normal holoenzyme in the oxidized and reduced form, respectively. Addition of chelator to holoenzyme did not lead to inactivation or monomerization. It is concluded, therefore, that Ca2+ has a dual role in this enzyme, being required for dimerization of the subunits as well as for functionalization of the bound PQQ, and that it is more firmly attached to the holoenzyme than to the apoenzyme.
Olsthoorn
A J
AJ
Department of Microbiology and Enzymology, Delft University of Technology, Julianalaan, The Netherlands.
Duine
J A
JA
D016954:Acinetobacter calcoaceticus / Q000201:enzymology*; D001051:Apoenzymes / Q000096:biosynthesis*; D042843:Glucose 1-Dehydrogenase; D005948:Glucose Dehydrogenases / Q000096:biosynthesis*; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins; D012995:Solubility
D001051:Apoenzymes; D011994:Recombinant Proteins; D005948:Glucose Dehydrogenases; D042843:Glucose 1-Dehydrogenase