Identification of the dimerization domain of dehalogenase IVa of Burkholderia cepacia MBA4.
Haloacid dehalogenases are enzymes that catalyze the hydrolytic removal of halogens from haloalkanoic acids. Dehalogenase IVa (DehIVa) from Burkholderia cepacia MBA4 and dehalogenase CI (DehCI) from Pseudomonas sp. strain CBS3 exhibit 68% identity. Despite their similarity DehIVa is a dimeric enzyme while DehCI is a monomer. In this work, we describe the identification of the domain that confers the dimerization function of DehIVa. Recombinant DNA molecules were constructed by fusion of the respective dehalogenase genes hdlIVa and dehCI. When amino acids 73 to 89 of DehCI were replaced by amino acids 74 to 90 of DehIVa, the recombinant molecule migrated like that of DehIVa in a nondenaturing activity-stained gel. Similarly, when residues 73 to 89 of DehIVa were replaced by the corresponding residues of DehCI, the chimera migrated as a monomer. These 17 amino acid changes were able to determine the aggregation states of the molecules. The retention of the catalytic function in these chimeras indicated that the overall folding of these proteins was not affected. Site-directed mutagenesis on hdlIVa however indicated that amino acids Phe58, Thr65, Leu78, and Phe92 of DehIVa are also important for the aggregation state of the protein. This indicates that the 17 residues are not sufficient for the dimerization of the protein.
Tsang
J S
JS
Molecular Microbiology Laboratory, Department of Botany, The University of Hong Kong, China. jshtsang@hkucc.hku.hk
Pang
B C
BC
D015153:Blotting, Western; D016956:Burkholderia cepacia / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D011549:Pseudomonas / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
D011993:Recombinant Fusion Proteins; D006867:Hydrolases; C034507:2-haloacid dehalogenase