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

Abstract

BackgroundMaltogenic amylases belong to a subclass of cyclodextrin-hydrolyzing enzymes and hydrolyze cyclodextrins more efficiently than starch unlike typical α-amylases. Several bacterial malto-genic amylases with temperature optima of 40–60°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 FindingsThe recombinant monomeric and dimeric forms of maltogenic α-amylase (Gt-Mamy) of the extremely thermophilic bacterium Geobacillus thermoleovorans are of 72.5 and 145 kDa, which are active optimally at 80°C. Extreme thermostability of this enzyme has been explained by analyzing far-UV CD spectra. Dimerization increases T1/2 of Gt-Mamy from 8.2 h to 12.63 h at 90°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 (Vmax/Km) 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 β-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 (α/β)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/SignificanceN-terminal domain controls enthalpy-driven thermostabilization, substrate-binding affinity and transglycosylation activity of Gt-Mamy by homodimer formation.

Genetic and Biochemical Characterization of a Novel Thermostable Cyclomaltodextrinase From Anoxybacillus flavithermus

“…In this study, command lines of MODELLER program were used for construction of a 3D dimeric form of CDase. Structural examination of the modelled enzyme confirmed that the central domain in the CDase has the functional property as described by Fritzsche et al and Unban et al Structural features of the enzyme show that the position of active sites in the dimeric form of the enzyme allows small‐sized substrates better entrance to the reaction site …”

“…The ratio of k cat / K m as an indication of substrate specificity for different substrates increases as α‐CD>β‐CD>γ‐CD. The resulting data of this study show that the recombinant CDase had more specificity toward α‐CD . Similar to CDase‐ZNU, α‐CD is the specific substrate for the CDase isolated from A. flavithermus (AAX29991.1) .…”

“…The resulting data of this study show that the recombinant CDase had more specificity toward α-CD. [53] Similar to CDase-ZNU, α-CD is the specific substrate for the CDase isolated from A. flavithermus (AAX29991.1). [44] The CDases from E. faecium K-1, [47] Bacillus sphaericus ATCC7055 [54] and T. ethanolicus 39E [10] also had more specificity for α-CD as compared to β-CD and γ-CD, while the CDases from Bacillus sphaericus E-244, [51] Bacillus spp.…”

“…Structural examination of the modelled enzyme confirmed that the central domain in the CDase has the functional property as described by Fritzsche et al [12] and Unban et al [47] Structural features of the www.advancedsciencenews.com www.starch-journal.com enzyme show that the position of active sites in the dimeric form of the enzyme allows small-sized substrates better entrance to the reaction site. [53] It has been reported that metal ions play a significant role in the activity of α-amylase family. [58] Investigating the effect of representative metal ions on the activity of the enzyme showed that the presence of some metal ions led to the decrease of the enzyme activity.…”

Characterization and gene cloning of a maltotriose-forming exo-amylase from Kitasatospora sp. MK-1785

“…G3Amy inefficiently hydrolyzed cyclodextrins (Table 2) as compared to that of other exo-α-amylases such as Geobacillus maltogenic amylases (Mehta and Satyanarayana 2013;Nasrollahi et al 2013). This indicated that the nonreducing end of a substrate was helpful to recognize the substrate, but not essential for hydrolysis by G3Amy unlike β-amylases.…”