scholarly journals Engineering and Directed Evolution of a Ca2+Binding Site A-Deficient AprE Mutant Reveal an Essential Contribution of the Loop Leu75–Leu82to Enzyme Activity

2009 ◽  
Vol 2009 ◽  
pp. 1-7
Author(s):  
Eliel R. Romero-García ◽  
Alfredo Téllez-Valencia ◽  
María F. Trujillo ◽  
José G. Sampedro ◽  
Hugo Nájera ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Binding Site ◽  
Directed Evolution ◽  
Calcium Binding ◽  
Thermal Inactivation ◽  
Fluorescence Analysis ◽  
Mutant Gene ◽  
The Novel ◽  
Calcium Binding Site ◽  
Essential Contribution

AnaprEmutant fromB. subtilis168 lacking the connecting loopLeu75–Leu82which is predicted to encode aCa2+binding site was constructed. Expression of the mutant gene (aprEΔLeu75–Leu82) producedB. subtiliscolonies lacking protease activity. Intrinsic fluorescence analysis revealed spectral differences between wild-type AprE and AprEΔL75–L82. An AprEΔL75–L82variant with reestablished enzyme activity was selected by directed evolution. The novel mutationsThr66Met/Gly102Asp located in positions which are predicted to be important for catalytic activity were identified in this variant. Although these mutations restored hydrolysis, they had no effect with respect to thermal inactivation of AprEΔL75–L82 T66M G102D. These results support the proposal that in addition to function as a calcium binding site, the loop that connectsβ-sheet e3 withα-helix c plays a structural role on enzyme activity of AprE fromB. subtilis168.

scholarly journals A disulfide bridge in the calcium binding site of a polyester hydrolase increases its thermal stability and activity against polyethylene terephthalate

FEBS Open Bio ◽  
2016 ◽  
Vol 6 (5) ◽  
pp. 425-432 ◽  
Author(s):  
Johannes Then ◽  
Ren Wei ◽  
Thorsten Oeser ◽  
André Gerdts ◽  
Juliane Schmidt ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Binding Site ◽  
Polyethylene Terephthalate ◽  
Calcium Binding ◽  
Disulfide Bridge ◽  
Calcium Binding Site

Calcium-Binding Site β2, Adjacent to the “b” Polymerization Site, Modulates Lateral Aggregation of Protofibrils during Fibrin Polymerization†,‡

Biochemistry ◽  
2004 ◽  
Vol 43 (9) ◽  
pp. 2475-2483 ◽  
Author(s):  
Michael S. Kostelansky ◽  
Karim C. Lounes ◽  
Li Fang Ping ◽  
Sarah K. Dickerson ◽  
Oleg V. Gorkun ◽  
...  
Keyword(s):  
Binding Site ◽  
Calcium Binding ◽  
Calcium Binding Site ◽  
Fibrin Polymerization

scholarly journals The calcium-binding site of human glutamate carboxypeptidase II is critical for dimerization, thermal stability, and enzymatic activity

2018 ◽  
Vol 27 (9) ◽  
pp. 1575-1584 ◽  
Author(s):  
Jakub Ptacek ◽  
Jana Nedvedova ◽  
Michal Navratil ◽  
Barbora Havlinova ◽  
Jan Konvalinka ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Enzymatic Activity ◽  
Binding Site ◽  
Calcium Binding ◽  
Calcium Binding Site ◽  
Glutamate Carboxypeptidase Ii ◽  
Glutamate Carboxypeptidase

scholarly journals Structural and Functional Characterization of Three Novel Fungal Amylases with Enhanced Stability and pH Tolerance

2019 ◽  
Vol 20 (19) ◽  
pp. 4902 ◽  
Author(s):  
Christian Roth ◽  
Olga V. Moroz ◽  
Johan P. Turkenburg ◽  
Elena Blagova ◽  
Jitka Waterman ◽  
...  
Keyword(s):  
Binding Site ◽  
Calcium Binding ◽  
Functional Characterization ◽  
Industrial Applications ◽  
Glycoside Hydrolases ◽  
Industrial Processes ◽  
Calcium Binding Site ◽  
Ph Tolerance ◽  
Speed Up ◽  
Binding Cleft

Amylases are probably the best studied glycoside hydrolases and have a huge biotechnological value for industrial processes on starch. Multiple amylases from fungi and microbes are currently in use. Whereas bacterial amylases are well suited for many industrial processes due to their high stability, fungal amylases are recognized as safe and are preferred in the food industry, although they lack the pH tolerance and stability of their bacterial counterparts. Here, we describe three amylases, two of which have a broad pH spectrum extending to pH 8 and higher stability well suited for a broad set of industrial applications. These enzymes have the characteristic GH13 α-amylase fold with a central (β/α)8-domain, an insertion domain with the canonical calcium binding site and a C-terminal β-sandwich domain. The active site was identified based on the binding of the inhibitor acarbose in form of a transglycosylation product, in the amylases from Thamnidium elegans and Cordyceps farinosa. The three amylases have shortened loops flanking the nonreducing end of the substrate binding cleft, creating a more open crevice. Moreover, a potential novel binding site in the C-terminal domain of the Cordyceps enzyme was identified, which might be part of a starch interaction site. In addition, Cordyceps farinosa amylase presented a successful example of using the microseed matrix screening technique to significantly speed-up crystallization.

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