Identification of the dimerization domain of dehalogenase IVa of Burkholderia cepacia MBA4

Abstract

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.

FIG. 1

Various chimeric dehalogenases and their electrophoretic properties. (a) Schematic representation of various constructs. The open box indicates the DehIVa portion, while the shaded box represents the DehCI region. The numbers represent the numbering of the amino acids of the corresponding enzyme. The last column indicates the dimeric (D) or monomeric (M) nature of the chimera. (b) Autoradiograph of an SDS-PAGE gel analyzing the in vitro-produced proteins. The proteins were synthesized in the presence of [³⁵S]methionine and analyzed on a 12.5% denaturing gel. (c) Nondenaturing activity-stained gel of various native and in vitro-produced proteins. The activities of the dehalogenases and derivatives were visualized using MCA as the substrate. (d) Autoradiograph of the activity-stained gel shown in panel c. (b to d) Lanes: 1, CE of B. cepacia MBA4; 2, CE of E. coli producing DehCI; 3 to 9, in vitro-produced HKU221, HKU252, HKU272, HKU271, HKU273, HKU275, and HKU281, respectively; 10, in vitro-produced mock lysate.

FIG. 1

Various chimeric dehalogenases and their electrophoretic properties. (a) Schematic representation of various constructs. The open box indicates the DehIVa portion, while the shaded box represents the DehCI region. The numbers represent the numbering of the amino acids of the corresponding enzyme. The last column indicates the dimeric (D) or monomeric (M) nature of the chimera. (b) Autoradiograph of an SDS-PAGE gel analyzing the in vitro-produced proteins. The proteins were synthesized in the presence of [³⁵S]methionine and analyzed on a 12.5% denaturing gel. (c) Nondenaturing activity-stained gel of various native and in vitro-produced proteins. The activities of the dehalogenases and derivatives were visualized using MCA as the substrate. (d) Autoradiograph of the activity-stained gel shown in panel c. (b to d) Lanes: 1, CE of B. cepacia MBA4; 2, CE of E. coli producing DehCI; 3 to 9, in vitro-produced HKU221, HKU252, HKU272, HKU271, HKU273, HKU275, and HKU281, respectively; 10, in vitro-produced mock lysate.

FIG. 2

Activity-stained gel of various native and chimeric dehalogenases. Lanes 1, cell extract of B. cepacia MBA4; 2, cell-free extract of E. coli producing DehCI; 3–7, in vitro-produced (IVP) HKU221, HKU252, HKU285, and HKU284, respectively; 8, IVP mock lysate.

FIG. 3

Western blot analysis of DehIVa derivatives in nondenaturing gel. Each of the recombinant molecules carries a FLAG leader peptide. A 10-μg aliquot of total cellular protein was loaded in each lane. Lanes 1 to 9, in vivo-expressed protein HKU355 (M55L), HKU357 (Y58F), HKU358 (F61Y), HKU359 (T65A), HKU360 (A68T), HKU361 (L69M), HKU362 (L78M), HKU363 (Y92F), and HKU364 (L108M).

FIG. 4

Relative molecular weight of the proteins used in this study. A Sephacryl S200 HR column was calibrated with protein standards of known molecular weight. The standard curve was plotted with the molecular weight (in thousands) versus the V_e/V_o ratio. V_e is the elution volume and V_o is the void volume as determined by blue dextran. DehIVa, HKU221, HKU272, HKU273, HKU275, HKU281, HKU285, and HKU356 are dimers and eluted around 46K (within the open circle). DehCI, HKU252, HKU271, HKU284, HKU357, and HKU359 are monomers and eluted around 30K (within the open square). HKU362 and HKU363 contain both dimer and monomer and were found eluted around the 46K and the 30K regions.