T. Chrobáková1, Z. Prokop1, Y. Sato2, M. Monincová1, A. Jesenská1, L. Grodecká1, T. Senda3, Y. Nagata2 and J. Damborský1


1 Loschmidt Laboratories, Faculty of Science, Masaryk University, Kamenice 5/A4,

625 00 Brno, Czech Republic

2 Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Sendai 980-8577, Japan

3 Biological Information Research Center, National Institute of Advanced Industrial Science and Technology, 2-41-6 Aomi, Koto-ku, Tokyo 135-0064, Japan


Enantioselective enzymes are useful as catalyst for biosynthesis of various organic compounds. Members of the haloalkane dehalogenase family (EC, which belong to superfamily of α/β-hydrolases, produce alcohols during the dehalogenating reaction. If dehalogenases are enantioselective, they could catalyse synthesis of several alcohols important for pharmaceutical, food and agricultural industry. However, only DbjA from Bradyrhizobium japonicum USDA110 [1] is showing significant enantioselectivity towards several types of substrates. The objective of this project is to extend this interesting property to other family members.  The most structurally related haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 was selected for mutagenesis.

Amino acids in the active site of DbjA, possibly determining enantioselectivity of this enzyme, were included in experimental design of reconstruction of enantioselective haloalkane dehalogenase DhaA. Residues were chosen based on sequence alignment of DbjA with DhaA. In addition to nine point substitutions (H105Q, W141F, P142A, F144A, G171R, A172V, K175G, C176G and V245A), the main difference is in the presence of the insertion loop (139HHTEVAEEQDH149) between the main and the cap domain of DbjA.

Seven rounds of mutagenesis were designed. At first, the mutant gene containing the loop-coding sequence was prepared by inverse polymerase chain reaction. Other mutations were cumulatively added using QuikChangeTM Site-Directed Mutagenesis Kit (Stratagene, USA) according to manufacturer’s instructions. Genes were amplified using specific complementary primers introducing substitutions into sequences. The genes carrying mutations were cloned into the pET21b expression vector (Novagen, USA) and expressed in Escherichia coli BL21 (DE3). Proteins were purified using HiTrapTM Chelating column with metal affinity resin (Amersham Biosciences, Germany). Their molecular weight and purity were checked by SDS-polyacrylamide electrophoresis. Folding of proteins was verified by circular dichroism (CD) spectroscopy. The dehalogenating activity of enzymes towards 1,3-dibromopropane and stereospecificity with the racemic mixture of the set of β-substituted aliphatic bromoalkanes and β-halogenated ester were tested by gas chromatography.

Six mutant genes of haloalkane dehalogenase DhaA were successfully constructed. All proteins were produced in a soluble form. The yields of protein variants except the first mutant were high. Four out of six mutants were active towards 1,3-dibromopropane. Activity data were consistent with the changes that were observed in CD spectra. Last two mutants with the most similar CD spectra to DhaA showed activity comparable to the wild type enzyme. Testing of mutants for enantioselectivity is in progress. The results of these experiments will be presented during the lecture.

1.    Y. Sato, M. Monincová, R. Chaloupková, Z. Prokop, Y. Ohtsubo, K. Minamisawa, M. Tsuda, J. Damborský & Y. Nagata, Appl. Environ. Microbiol., 71, (2005), 4372-4379.