Stabilization of quaternary structure of water-soluble quinoprotein glucose dehydrogenase.
Water-soluble quinoprotein glucose dehydrogenase (PQQGDH-B) is a dimeric enzyme whose application for glucose sensing is the focus of much attention. We attempted to increase the thermal stability of PQQGDH-B by introducing a disulfide bond at the dimer interface. The Ser residue at position 415 was selected for substitution with Cys, as structural information revealed that its side chains face each other at the dimer interface of PQQGDH-B. PQQGDH-B with Ser415Cys showed 30-fold greater thermal stability at 55 degrees C than did the wild-type enzyme without any decrease in catalytic activity. After incubation at 70 degrees C for 10 min, Ser415Cys retained 90% of the GDH activity of the wild-type enzyme. Disulfide bond formation between the mutant subunits was confirmed by analyses with sodium dodecylsulfate-polyacrylamide gel electrophoresis in the presence and absence of reductants. Our results indicate that the introduction of one Cys residue in each monomer of PQQGDH-B resulted in formation of a disulfide bond at the dimer interface and thus achieved a large increase in the thermal stability of the enzyme.
Igarashi
Satoshi
S
Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
Sode
Koji
K
D003198:Computer Simulation; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004789:Enzyme Activation; D004795:Enzyme Stability; D005948:Glucose Dehydrogenases / Q000737:chemistry* / Q000235:genetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry; D012995:Solubility; D013329:Structure-Activity Relationship; D013696:Temperature; D014867:Water / Q000737:chemistry*
D004220:Disulfides; D046911:Macromolecular Substances; D021122:Protein Subunits; D011994:Recombinant Proteins; D014867:Water; D005948:Glucose Dehydrogenases; C033955:glucose dehydrogenase (pyrroloquinoline-quinone)