Increasing stability of water-soluble PQQ glucose dehydrogenase by increasing hydrophobic interaction at dimeric interface

Abstract

Background: Water-soluble quinoprotein glucose dehydrogenase (PQQGDH-B) from Acinetobacter calcoaceticus has a great potential for application as a glucose sensor constituent. Because this enzyme shows no activity in its monomeric form, correct quaternary structure is essential for the formation of active enzyme. We have previously reported on the increasing of the stability of PQQGDH-B by preventing the subunit dissociation. Previous studies were based on decreasing the entropy of quaternary structure dissociation but not on increasing the interaction between the two subunits. We therefore attempted to introduce a hydrophobic interaction in the dimeric interface to increase the stability of PQQGDH-B.

Results: Amino acid residues Asn340 and Tyr418 face each other at the dimer interface of PQQGDH-B, however no interaction exists between their side chains. We simultaneously substituted Asn340 to Phe and Tyr418 to Phe or Ile, to create the two mutants Asn340Phe/Tyr418Phe and Asn340Phe/Tyr418Ile. Furthermore, residues Leu280, Val282 and Val342 form a hydrophobic region that faces, on the other subunit, residues Thr416 and Thr417, again without any specific interaction. We simultaneously substituted Thr416 and Thr417 to Val, to create the mutant Thr416Val/Thr417Val. The temperatures resulting in lose of half of the initial activity of the constructed mutants were increased by 3-4 degrees C higher over wild type. All mutants showed 2-fold higher thermal stability at 55 degrees C than the wild-type enzyme, without decreasing their catalytic activities. From the 3D models of all the mutant enzymes, the predicted binding energies were found to be significantly greater that in the wild-type enzyme, consistent with the increases in thermal stabilities.

Conclusions: We have achieved via site-directed mutagenesis the improvement of the thermal stability of PQQGDH-B by increasing the dimer interface interaction. Through rational design based on the quaternary structure of the enzyme, we selected residues located at the dimer interface that do not contribute to the intersubunit interaction. By substituting these residues to hydrophobic ones, the thermal stability of PQQGDH-B was increased without decreasing its catalytic activity.

Figure 1

The amino acid residues located at the dimer interface. The two subunits are represented in red and blue, respectively, using the RasMol molecular visualization software 26. The 19 amino acid residues at the interface are shown in space filling format, of which 8 residues (orange and light blue) are predicted not be involve in hydrogen bond formation, electrostatic interaction, or hydrophobic interaction at the interface.

Figure 2

Hydrophobicity of PQQGDH-B dimer interface. Hydrophobic regions are shown in green and hydrophilic regions are shown in blue. Residues that have been substituted in this study are indicated. The structural images were generated using Molecular Operating Environment.

Figure 3

Thermal stability of wild-type and mutant PQQGDH-Bs. Residual activity was measured at 25°C after 10-min incubations at different temperatures of the following protein samples (0.075 μg/mL): Wild-type ◆, Asn340Phe/Tyr418Phe □, Asn340Phe/Tyr418Ile △, and Thr416Val/Thr417Val ○.

Figure 4

Time course of thermal inactivation of wild-type and mutant PQQGDH-Bs. Protein samples (0.075 μg/mL) were incubated at 55°C and aliquots were taken at different times to measure residual activity. The analyzed samples contained the following PQQGDH-Bs: Wild-type ◆, Asn340Phe/Tyr418Phe □, Asn340Phe/Tyr418Ile △, Thr416Val/Thr417Val ○.

Figure 5

Hydrophobicity of mutant PQQGDH-B dimer interface. Hydrophobic regions are shown in green while hydrophilic regions are shown in blue. The interfaces shown are those of the Asn340Phe/Tyr418Phe (left), Asn340Phe/Tyr418Ile (middle), and Thr416Val/Thr417Val (right) mutants of PQQGDH-B, with their respective mutation sites circled. The structural images were generated using Molecular Operating Environment.