Identification of the Dimerization Domain of Dehalogenase IVa ofBurkholderia cepaciaMBA4

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.Dehalogenases are enzymes that remove halogen from the carbon moiety (6). Several dehalogenases have been purified and characterized since their first detection in bacteria. These dehalogenases were categorized according to their substrate specificities (28) and recently according to their phylogenetic relationships (8). Much attention has been drawn to the 2-haloacid dehalogenases or halidohydrolases. These are hydrolytic enzymes that cleave the halogen-carbon bond(s) in halogenated aliphatic acids, yielding hydroxy-or oxoalkanoic acids from a substrate with a mono-or disubstitution, respectively (7, 28). At least 11 2-haloacid dehalogenase genes have been isolated and sequenced. Comparative study on the amino acid sequences of these enzymes has exhibited 37 to 67% homology (17, 32). Among these enzymes three conserved motifs have been identified. These include residues 4 to 18 in motif 1, residues 105 to 123 in motif 2, and residues 139 to 194 in motif 3 (1). Motif 1 contains a highly conserved aspartate and a threonine, motif 2 contains a highly conserved hydroxy residue (serine or threonine), and motif 3 contains a highly conserved lysine and a pair of aspartates. These conserved motifs were expected to convey functions essential for catalysis. Site-directed mutagenesis had confirmed the role of these motifs in the activity of dehalogenase L-DEX-YL and dehalogenase IVa (DehIVa) (18; B. C. M. Pang and J. S. H. Tsang, unpublished data).Most dehalogenases were identified from microorganisms isolated from enrichment cultures using specific halogenated substrates (3, 27). These microorganisms are capable of utilizing the substrates as sole carbon and energy sources. Burkholderia cepacia MBA4, isola...

Isolation and characterization of monochloroacetic acid-degrading bacteria

“…However, previously, it was reported that DehCI of Pseudomonas sp. strain CBS3 had low degradation activity toward DCA, and that Hdl IVa of Burkholderia cepacia strain MBA4 could not degrade DCA (Tsang and Pang, 2000). Because the dehalogenase in our isolates has high degradation activity toward DCA, it may be a good resource for further investigation.…”

Evolutionary Genomics of the HAD Superfamily: Understanding the Structural Adaptations and Catalytic Diversity in a Superfamily of Phosphoesterases and Allied Enzymes

“…The dehr subfamily II shows even greater diversity in motif IV (e.g SSNxxD, SSxxxD and AAxxxD) with wide differences in the conservation of the acidic residues and is consistent with the acquisition of non-phosphate substrates such as haloalkanoic acids. 137,138 The enolase-phosphatase family of enzymes catalyzes the oxidative dephosphorylation (in combination with the enolase) of 2,3-diketo-1-phosphohexane to 2-keto-pentanoate in the latter steps of the methionine salvage pathway. 139,140 Members of the restricted bacterial Bcs3 family are fused to an Nterminal glycosyltransferase domain (Bcs3_Hinf in Figure 9) and might function as sugar phosphatases in the biosynthesis of capsular polysaccharides in certain pathogenic bacteria 141 (Table 1).…”

Isolation and Characterization of a Novel Haloacid Permease from Burkholderia cepacia MBA4

“…Deh4a exists as a dimeric protein of 45 kDa, composed of two identical subunits of 23 kDa each (27,45). The regions involved in the dimerization and the activity functions of the enzyme have also been elucidated (30,46). Deh4a is mainly found in the cytoplasm of B. cepacia MBA4, and no signature of any signal sequence has been identified.…”