Role of the buried halide-binding site of haloalkane dehalogenase DbeA
Daniel L.1, Chaloupkova R.1, Brezovsky J.1, Prudnikova
T.2, Rezacova P.3, Prokop Z.1, Mozga T.1, Koudelakova T.1,
Sato Y.4, Kuty M.2,5, Nagata Y.4,
Kuta Smatanova I.2,5 and
Damborsky J.1
1 Loschmidt Laboratories, Department of Experimental Biology
and Research Centre for Toxic Compounds in the Environment, Faculty of Science,
Masaryk University, Kamenice 5/A4, 625 00 Brno, Czech
Republic;
2South Bohemian Research
Center of Aquaculture and Biodiversity of Hydrocenoses
and School of Complex Systems, University of South Bohemia, Zamek
136, 373 33 Nove Hrady,
Czech Republic;
3Institute of Molecular
Genetics, Academy of Sciences of the Czech Republic, Videnska
1083, 142 20 Prague, Czech Republic
4, Czech Republic, 4Department
of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku
University, 2-1-1 Katahira, Sendai 980-8577, Japan;
5Institute of Nanobiology and Structural
Biology GCRC, Academy of Sciences of the Czech Republic, Zamek
136, 373 33 Nove Hrady,
Czech Republic
The crystal structure of haloalkane dehalogenase DbeA from Bradyrhizobium elkani USDA94 revealed the presence of a unique second halide-binding site for chlorides. A double mutant DbeA03 (I44L+Q102H), which should have the second binding site removed, was constructed and biochemically characterized. Molecular modeling was employed to prove successful removal of the second halide-binding site in the mutant and to study its role in the catalysis.
Comparison of calculated binding energies
of chloride ions bound at the second halide-binding site in DbeA03 and wild
type DbeA suggested the successful removal of the
second halide-binding site. The calculated difference in the binding energies
between wild type DbeA and DbeA03 was 8.7 ± 2.7 kcal.mol-1. This conclusion was confirmed
experimentally by stopped flow fluorescence measurement of chloride binding to
both enzymes. Obtained dissociation constant showed an order of magnitude
decrease in chloride binding affinity to DbeA03 compared to DbeA
wt. The effect of the second
halide-binding site on the catalysis was consequently probed by molecular
dynamic simulations at constant pH conditions. The pKa of the catalytic histidine in wild type DbeA
(pKa
= 7.1 ± 1.4) without chloride anion bound at the second halide-binding site was
comparable to pKa
in DbeA03 (pKa
= 7.3 ± 0.5) where the second halide-binding site was removed. In the case of wild
type DbeA with the chloride anion present, the pKa of
the catalytic histidine was significantly increased (pKa =
9.6 ± 0.8) making it a much stronger base. This effect is in agreement with transient
kinetic experiments revealing that the rate of hydrolysis was significantly
decreased by introduced mutations.
This study showed that the presence of the second halide-binding site in haloalkane dehalogenase DbeA significantly alters its catalytic properties. Thus, engineering of buried halide-binding sites into the protein core represents a novel strategy for the construction of novel catalysts.