Understanding Enzymes: Function, Design, Engineering and Analysis

0523217 - BTÚ 2020 RIV SG eng M - Část monografie knihy
Dohnálek, Jan - McAuley, K. E. - Brzozowski, A. M. - Ostergaard, P. R. - Svendsen, A. - Wilson, K. S.
Stabilization of enzymes by metal binding: structures of two alkalophilic Bacillus subtilases and analysis of the second metal-binding site of the subtilase family.
Understanding Enzymes: Function, Design, Engineering and Analysis. Singapore: Pan Stanford Publishing Pte. Ltd., 2016 - (Svendsen, A.), s. 203-265. ISBN 978-981-4669-32-0
Grant CEP: GA MŠMT(CZ) ED1.1.00/02.0109
Institucionální podpora: RVO:86652036
Klíčová slova: X-ray crystallography * enzyme structure * metal binding
Obor OECD: Biochemistry and molecular biology

The subtilases make up one of the most intensively studied families of enzymes. Subtilases have acted as paradigms for studies inter alia of kinetics and mechanism, 3D structure, protein–ligand interactions, stability to denaturation, complexes with naturally occurring polypeptide inhibitors, and site-directed mutagenesis. Bacillus subtilisins share several key features, such as a leader sequence to ensure transport from the cell, with the majority of the family possessing di- and/or monovalent metal-binding sites that contribute to stability. In the classical subtilisins there is a strong calcium-binding site and a second weak site that has an alternative sodium site close to it. Thus while the principle of using calcium as an extracellular stabilizing agent is exploited by all the extracellular subtilases, it has been independently lost or acquired at various points during their evolution. We report here the crystal structures of two subtilases from Bacillus sp. TY145 and from B. halmapalus, metal-stabilized serine proteases with biotechnological potential. In addition, a detailed analysis of metal binding in more than eighty similar enzymes is reported. The identity of the ion in the second binding site of subtilases – often misinterpreted – was correctly assigned in most cases. Interestingly, the roles of sodium and calcium had been incorrectly exchanged and misinterpreted. Our work sheds new light on many previous works on technologically applied subtilases.
Trvalý link: http://hdl.handle.net/11104/0307594