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Tasks:
crystallization of the mutant of haloalkane
dehalogenase enzyme
testing of protein crystals and following data collection
work with computer program for creating protein structure
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/-Hydrolase Superfamily
Subfamily
HLD-I
HLD-II
HLD-III
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Active site residues
(catalytic pentad)
Asn41
Trp107
Asp106
His272
Glu130
Reaction mechanism ofhaloalkane dehalogenases
with-bromoestersand -bromoalkanes. Enz-COO : active siteAsp.
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DhaA & DbjA
DhaA the most structurally related to DbjA
Several residues from the active site and tunnel of DhaA were
changed to ones corresponding to DbjA
The mutant DhaA12 is protein combining the nature of two parentsproteins
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PEG 4000 , 20%;
MESSodium salt, pH 6.0;
protein concentration 7 mg/ml;
Proportion 1:1
PEG 4000, 20%;
MESSodium salt, pH 6.0;
protein concentration 8 mg/ml;
Proportion 1:1
PEG 4000, 19%;
MESSodium salt, pH 6.2;
protein concentration 9.2 mg/ml;
Proportion 1:1
PEG 4000 , 19%;
MESSodium salt, pH 6.0;
protein concentration 8 mg/ml ;proportion 1:2
PEG 4000, 20%;
MESSodium salt, pH 6.1;
protein concentration 8 mg/ml;Proportion 1:1
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Crystals belonged to the orthorhombic space groupP212121
X 57
0.078mm
Diffracted to the resolution of 1.78
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active site
Loop (unique sequence )
W141FF144A
P142A
V245A
G171R
A172V
C176G
K175G
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Alignment ofDhaA&DhaA12
structures
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Asn41
Trp107
Asp106
His272
Glu 130
Active site
electron density catalytic pentad
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mutant protein DhaA12 was crystallized by standard vapor
diffusion technique sitting drop method
data processing was done. Structure of DhaA04 was used formolecular replacement as a template
the structure was refined using SHELXL and manual building
parts were preformed in COOT software
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Institute of Physical Biology University of South Bohemia, Ceske Budejovice
-Alena Stsiapanava, MSc.
- Ivana Kuta-Smatanova, PhD.
Loschmidt Laboratories, Faculty of Science, Masaryk University, Brno
- Prof. Jiri Damborsky, Dr.- Radka Chaloupkova, PhD.
Institute of Biochemistry, Luebeck University
- Jeroen Mesters, Dr.
- Prof. R. Hilgenfeld, Dr.
BESSYII booster synchrotron, Berlin
- Uwe Mueller , Dr.
- Manfred S. Weiss, Dr.
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Thank you for attention