CRYSTALLIZATION OF THREE MUTANTS DERIVED FROM HALOALKANE DEHALOGENASE DHAA OF Rhodococcus rhodochrous NCIMB 13064

Alena Stsiapanava¹, Tana Koudelakova³, Lucie Grodecka³_, Jiri Damborsky³, and Ivana Kuta Smatanova^1,2

¹Institute of Physical Biology University of South Bohemia Ceske Budejovice, Zamek 136, 373 33 Nove Hrady, Czech Republic

²Institute of Systems Biology and Ecology Academy of Science of the Czech Republic, Zamek 136, 373 33 Nove Hrady, Czech Republic

³Loschmidt Laboratories, Faculty of Science, Masaryk University, Kamenice 5/A4, 62500 Brno, Czech Republic

stepanova@greentech.cz

Microbial growth on halogenated substrates requires the production of catabolitic enzymes that cleave carbon-halogen bonds. Such enzymes are commonly called dehalogenases [1]. Haloalkane dehalogenases (EC 3.8.1.5) are enzymes that belong to the α/β-hydrolase fold superfamily [2]. These microbial enzymes catalyze the hydrolysis of haloalkanes to the corresponding alcohol, halide, and a hydrogen ion. From this point of view haloalkane dehalogenases are promising bioremediation and biocatalytic agents [3].

Wild-type DhaA was isolated from bacterium Rhodococcus rhodochrous NCIMB 13064 [4]. Derived mutant enzymes DhaA04, DhaA14 and DhaA15 were constructed to reveal importance of product transporting pathways (tunnels) in DhaA for its enzymatic activity. Our project is aimed to produce crystals of haloalkane dehalogenases DhaA04, DhaA14 and DhaA15 purified mutants in efficient quality for diffraction experiments and finally compare results with known structure of wild-type DhaA [3].

Standard vapor diffusion technique has been used for searching and optimization of crystallization conditions. Crystallization experiments have been performed in Hampton Research Linbro and Cryschem plates (Hampton Research, CA, USA) as well as in Emerald BioStructures CombiClover Crystallization Plate (EBS plate, Emerald BioStructures, WA, USA) using commercial crystallization kits such as Crystal Screen Lite and Crystal Screen of Hampton Research, and Clear Strategy Screen 1 of Molecular Dimensions Limited (MDL, Suffolk, UK) and also using home-made solutions.

Сrystallization experiments with all enzyme mutants are in the progress.

1. Dick B Janssen, Frens Pries, and Jan R. van der Ploeg: Genetics and biochemistry of dehalogenating enzymes. Microbiology, 48 (1994) 163-191.

2. Dick B Janssen: Evolving Haloalkane Dehalogenases. Current Opinion in Chemical Biology, 8 (2004) 150‑159

3. Janet Newman, Thomas S. Peat, Ruth Richard, Lynn Kan, Paul E. Swanson, Joseph A. Affholter, Ian H. Holmes, John F. Schindler, Clifford J. Unkefer and Thomas C. Terwilliger: Haloalkane Dehalogenases: Structure of a Rhodococcus Enzyme. Biochemistry, 38 (1999) 16105‑16114

4. Anna N. Kulakova, Michael J. Larkin and Leonid A. Kulakov: The plasmid-located haloalkane dehalogenase gene from Rhodococcus rhodochrous NCIMB 13064. Microbiology, 143 (1997) 109–115

This work is supported by the Ministry of Education of the Czech Republic (MSM6007665808 and LC06010) and by the Academy of Sciences of the Czech Republic (AVOZ60870520).