Maryna Lahoda, Mgr.

Institute of Physical Biology University of South Bohemia Ceske Budejovice,

Zamek 136, 373 33 Nove Hrady, Czech Republic
 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

 α/β-Hydrolase Superfamily

Subfamily

HLD-I

HLD-II

HLD-III
 α/β-Hydrolase Superfamily

Subfamily HLD-II

 LinB from Sphingobium japonicum,  show excellent

enantioselectivity for
α-bromoesters
 DhaA from Rhodococcus sp.

 DbjA from Bradyrhizobium japonicum

 identical catalytic pentad:
(showed high enantioselectivity with two β- Asp-His-Glu-Asn-Trp
bromoalkanes)
 Asn41

His272

Trp107
Asp106

Glu130
Reaction mechanism of haloalkane dehalogenases
with α-bromoesters
and β-bromoalkanes. Enz-COO : active site Asp.
–
Active site residues
(catalytic pentad)
 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 parents

proteins
 PEG 4000 , 20%; PEG 4000, 20%; PEG 4000, 19%;
MES Sodium salt, pH 6.0; MES Sodium salt, pH 6.0; MES Sodium salt, pH 6.2;
protein concentration 7 mg/ml; protein concentration 8 mg/ml; protein concentration 9.2 mg/ml;
Proportion 1:1 Proportion 1:1 Proportion 1:1

PEG 4000 , 19%; PEG 4000, 20%;

MES Sodium salt, pH 6.0; MES Sodium salt, pH 6.1;
protein concentration 8 mg/ml ; protein concentration 8 mg/ml;
proportion 1:2 Proportion 1:1
 0 .3
25 X 57
mm

0.078mm

 Crystals belonged to the orthorhombic space group P212121

 Diffracted to the resolution of 1.78 Å

 F144A active site
W141F
G171R
P142A

Loop (unique sequence )

A172V

V245A
K175G

C176G
 D H)
EEQ
A
T EV
(HH

Alignment of DhaA&DhaA12
structures
 Active site
Asn41

Trp107 His272

Asp106

Glu 130

electron density catalytic pentad

mutant protein DhaA12 was crystallized by standard vapor –
diffusion technique sitting – drop method

data processing was done. Structure of DhaA04 was used for

molecular replacement as a template

 the structure was refined using SHELXL and manual building

parts were preformed in COOT software
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.

BESSY–II booster synchrotron, Berlin

- Uwe Mueller , Dr.
- Manfred S. Weiss, Dr.
Thank you for attention