Dimerization Mediates Thermo-Adaptation, Substrate Affinity and Transglycosylation in a Highly Thermostable Maltogenic Amylase of Geobacillus thermoleovorans

Background: Maltogenic amylases belong to a subclass of cyclodextrin-hydrolyzing enzymes and hydrolyze cyclodextrins more efficiently than starch unlike typical alpha-amylases. Several bacterial malto-genic amylases with temperature optima of 40-60 degrees C have been previously characterized. The thermo-adaption, substrate preferences and transglycosylation aspects of extremely thermostable bacterial maltogenic amylases have not yet been reported. Methodology/Principal Findings: The recombinant monomeric and dimeric forms of maltogenic a-amylase (Gt-Mamy) of the extremely thermophilic bacterium Geobacillus thermoleovorans are of 72.5 and 145 kDa, which are active optimally at 80 degrees C. Extreme thermostability of this enzyme has been explained by analyzing far-UV CD spectra. Dimerization increases T-1/2 of Gt-Mamy from 8.2 h to 12.63 h at 90 degrees C and mediates its enthalpy-driven conformational thermostabilization. Furthermore, dime-rization regulates preferential substrate binding of the enzyme. The substrate preference switching of Gt-Mamy upon dimerization has been confirmed from the substrate-binding affinities of the enzyme for various high and low molecular weight substrates. There is an alteration in Km and substrate hydrolysis efficiency (V-max/K-m) of the enzyme (for cyclodex-trins/starch) upon dimerization. N-terminal truncation indicated the role of N-terminal 128 amino acids in the thermostabilization and modulation of substrate-binding affinity. This has been confirmed by molecular docking of beta-cyclodextrin to Gt-Mamy that indicated the requirement of homodimer formation by the interaction of a few N-terminal residues of chain A with the catalytic residues of (alpha/beta)(8) barrel of chain B and vice-versa for stable cyclodextrin binding. Site directed mutagenesis provided evidence for the role of N-terminal D109 at the dimeric interface in substrate affinity modulation and thermostabilization. The dimeric Gt-Mamy transglycosylates hydrolytic products of G4/G5 and acarbose, while the truncated form does not because of the lack of extra sugar-binding space formed due to dimerization. Conclusion/Significance: N-terminal domain controls enthalpy-driven thermostabilization, substrate-binding affinity and transglycosylation activity of Gt-Mamy by homodimer formation.