HotSpot Wizard: web server for identification of mutagenesis hot spots in enzyme structures
A. Pavelka$, E. Chovancová, J.
Damborský
Loschmidt Laboratories, Institute of
Experimental Biology and National Centre for Biomolecular Research
Faculty of Science, Masaryk University, Kamenice 5/A4, 625 00 Brno, Czech
Republic
$Email: tonda@chemi.muni.cz
HotSpot Wizard is a web server for
identification of hot spots for rational engineering of substrate specificity,
activity and enantioselectivity of enzymes. Rational design usually requires
considerable knowledge of structure-function relationships of a target protein.
Mutations targeting the residues located in the active sites or lining the
access tunnels, have a better chance to produce enzyme variants with novel
catalytic properties, than mutations localized in other parts of the protein
structure. On the other hand, replacement of the amino acid residues that are
highly conserved through evolution due to their essential role for the
structural stability or functionality, may lead to the loss of enzymatic
activity. Mutagenesis of variable sites, which contributes to the substrate
binding, transition state stabilization or product release, represents a useful
concept in protein engineering [1]. To follow this concept, the users need to
have experiences with analyses of protein sequences and structures.
Furthermore, these analyses are time-consuming even for experienced users.
HotSpot Wizard automates and speeds-up the
analysis by integration of structural and evolutionary information obtained
from selected bioinformatics databases and tools. HotSpot Wizard requires a
structure of the query protein in PDB format and an e-mail address as the only
obligatory inputs. In the first step, Catalytic Site Atlas [2] and UniProt [3]
databases are used to determine the residues indispensable for enzyme function.
HotSpot Wizard then searches for potentially important residues by CASTp [4],
identifying the active site pocket, and by CAVER [5, 6], calculating
tunnels connecting buried cavities with the outside solvent. Finally,
evolutionary conservation of individual positions in the query structure is
estimated by ConSurf [7] from the multiple sequence alignment of closely
related proteins. In the output, HotSpot Wizard lists residues ordered by
predicted suitability for mutagenesis together with information on their
conservation level, potential structural and functional importance, available
mutagenesis data and existing sequence variants. Results are mapped on the
enzyme structure and can be visualized directly in a web browser using Jmol [8]
or downloaded to the local computer as input for PyMOL [9] or text files.
The primary application of HotSpot Wizard
is in the rational design of the hot spots for site directed mutagenesis or
focused directed evolution. Alternatively, HotSpot Wizard can serve as a
structure annotation tool. Altogether, using HotSpot Wizard one can perform
several structural and evolutionary analyses at once with minimal demands on a
user, making this server potentially useful for experimentalists with no prior
knowledge of structural and bioinformatics analyses.
HotSpot Wizard is freely available at http://loschmidt.chemi.muni.cz/hotspotwizard/.
References
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