EVOLUTION OF HALOALKANE DEHALOGENASE PROTEIN FAMILY
E.Chovancová1$, J. Kosinski2, J. M. Bujnicki2 and J. Damborský1
1Loschmidt Laboratories, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, CZ
2Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, ul. Ks. Trojdena 4, 02-109 Warsaw, PL
Enzymes from the haloalkane dehalogenase protein family (EC 18.104.22.168) play an important role in bioremediation processes for their capability to hydrolyze haloorganic compounds. Currently, this family includes 14 experimentally characterized enzymes with proven dehalogenation activity. In addition to them, many putative members of this family can be found in sequence databases. In this study, we have used phylogenetic approach to assess the origin and evolution of haloalkane dehalogenases. Knowledge of the evolutionary history of haloalkane dehalogenases and related protein families should provide basis to understand the evolution of enzymatic activities and structure‑function relationships.
Over 3000 protein sequences, including haloalkane dehalogenases and their homologs, were identified through PSI-BLAST database searches . Obtained sequences were clustered using the program CLANS . Sequences from the cluster of haloalkane dehalogenases were aligned and used for phylogenetic reconstructions by maximum‑likelihood  and neighbor‑joining  methods. Various evolutionary models with different parameters were tested. For rooting of resulting trees, three alternative outgroups were used. Phylogenetic trees from all analyses were compared in terms of tree topology and placement of the root. Statistical techniques including four‑cluster likelihood mapping  were further used to test phylogenetic hypotheses.
Phylogenetic analysis of haloalkane dehalogenases indicated that the members of this family comprise three subfamilies designated HAD‑I, HAD‑II and HAD‑III. Most of biochemically characterized haloalkane dehalogenases (9 of 14) belong to the subfamily HAD‑II. Calculations performed under various conditions did not show significant differences in the results. Sister‑group relationship was suggested for subfamilies HAD‑I and HAD‑III, while HAD‑II subfamily appeared to be more distantly related. Preferential grouping of HAD‑I and HAD‑III subfamilies was indicated also by four‑cluster likelihood mapping. Based on the results, hypothesis about evolutionary history of haloalkane dehalogenases was proposed. Results of the analysis enabled identification of new family members and subsequent phylogenetic classification of the whole family. Furthermore, evolutionary studies of haloalkane dehalogenases will be useful for determination of conserved regions. Altogether, results will lead to improved theoretical predictions of functional, biochemical and structural properties of novel proteins. Putative haloalkane dehalogenases with potentially interesting properties will be cloned and biochemically characterized. This may lead to acquisition of proteins with novel characteristics suitable for practical applications.
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This work was supported by The European Commission 5th Framework Programme project ”Center of Excellence in Molecular Bio-Medicine Contract no: QLK6-CT-2002-90363” and grant from the Czech Ministry of Education no. MSM0021622412-3.