Cooperative Effect of Two Surface Amino Acid Mutations (Q252L and E170K) in Glucose Dehydrogenase from Bacillus megaterium IWG3 on Stabilization of Its Oligomeric State

Abstract

. This mutant had two amino acid substitutions, Q252L and E170K. In the present study, we characterized three GlcDH mutants harboring the substitutions Q252L, E170K, and Q252L/E170K under low-salt conditions. The GlcDH mutant harboring two substitutions, Q252L/E170K, was stable, but mutants harboring a single substitution, either Q252L or E170K, were unstable at an alkaline pH. Gel filtration chromatography analyses demonstrated that the oligomeric state of the Q252/E170K enzyme was stable, while the tetramers of the enzymes harboring a single substitution (Q252L or E170K) dissociated into dimers at an alkaline pH. These results indicated that the Q252L and E170K substitutions synergistically strengthened the interaction at the dimer-dimer interface. The crystal structure of the E170K/Q252L mutant, determined at 2.0-Å resolution, showed that residues 170 and 252 are located in a hydrophobic cavity at the subunit-subunit interface. We concluded that these residues in the wild-type enzyme have thermodynamically unfavorable effects, while the Q252L and E170K substitutions increase the subunit-subunit interactions by stabilizing the hydrophobic cavity.

Development of a Thermostable Glucose Dehydrogenase by a Structure‐Guided Consensus Concept

“…As in the case of single variants, the amino acid changes Q252L and E170A C H T U N G T R E N N U N G (K or R) in combined mutations of B. subtilis GDH contribute significantly to stabilization while the others (i.e., P45A, N46E, F155Y, V227A, W230F) make minor contributions, resulting in a plateau of stabilization for Q252L/ E170A C H T U N G T R E N N U N G (K or R) variants of GDH that does not seem to be overcome with additional mutations. The role of Q252L and E170A C H T U N G T R E N N U N G (K or R) for GDH stabilization is in agreement with the findings of Baik et al, [22,30] who discovered the importance of residues Q252L and E170K in the stability of GDH. Assisted by crystal structures from the wild-type and mutant GDHs from Bacillus megaterium IWG3, Baik et al determined that a cooperative effect between Q252L and E170K stabilizes the tetramer structure by strengthening the hydrophobic interactions within the interface [30] -significant, as GDH has been demonstrated to be an obligate tetramer.…”

“…The role of Q252L and E170A C H T U N G T R E N N U N G (K or R) for GDH stabilization is in agreement with the findings of Baik et al, [22,30] who discovered the importance of residues Q252L and E170K in the stability of GDH. Assisted by crystal structures from the wild-type and mutant GDHs from Bacillus megaterium IWG3, Baik et al determined that a cooperative effect between Q252L and E170K stabilizes the tetramer structure by strengthening the hydrophobic interactions within the interface [30] -significant, as GDH has been demonstrated to be an obligate tetramer. We surmise that E170R has a similar structural effect on GDH.…”

“…These criteria should be modified on the basis of available knowledge and data relating to a specific protein. For example, quaternary structure integrity is critical for activity and stability of GDH, [19,30] as well as many alcohol dehydrogenases. [31] Thus, strengthening of subunit-subunit interactions is critical for stabilization.…”

Crystal structure of a carbonyl reductase from Candida parapsilosis with anti‐Prelog stereospecificity

“…The pETSCR plasmid was used as a template to generate recombinant plasmids coding for SCR mutants. Site-directed mutagenesis was performed using standard protocols (Baik et al 2005). The mutations were verified by DNA sequencing.…”

Amperometric Biosensor Based on Glucose Dehydrogenase and Plasma-polymerized Thin Films

“…Since one dimension of GDH protein was ca. 10 nm from the crystallographic data, 23 the immobilized GDH array on PPF surface was probably covered with the 9-nm-and 18-nm-thickness PPF-coatings. 24 From Figs.…”