OF ENANTIOSELECTIVE HALOALKANE DEHALOGENASE BY CUMULATIVE MUTAGENESIS
Chrobáková1, Z. Prokop1, Y. Sato2, M.
Monincová1, A. Jesenská1, L. Grodecká1, T. Senda3, Y. Nagata2 and J. Damborský1
1 Loschmidt Laboratories, Faculty of
2 Department of Environmental Life
Biological Information Research Center, National
enzymes are useful as catalyst for biosynthesis of various organic compounds.
Members of the haloalkane dehalogenase family (EC 220.127.116.11), 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  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.
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 (
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.