title: Sec-dependent and Sec-independent translocation of haloacid dehalogenase Chd1 of Burkholderia cepacia MBA4 in Escherichia coli.
abstract: 2-Haloacid dehalogenases are hydrolytic enzymes that cleave the halogen-carbon bond(s) in haloalkanoic acids. We have previously isolated a cryptic haloacid dehalogenase gene from Burkholderia cepacia MBA4 and expressed it in Escherichia coli. This recombinant protein is unusual in having a long leader sequence, a property of periplasmic enzymes. In this paper, we report the functional role of this leader sequence. Western blot analyses showed that Chd1 is translocated to the periplasm. The results on the expression of Chd1 in the presence of sodium azide suggested the cleavage of the leader to be Sec-dependent. Chimeras of Chd1 and green fluorescent protein demonstrated that the leader sequence is fully functional in translocating the fusion protein to the periplasm. The expression of the chimeras in Sec mutants supported the Sec-dependent translocation. Surprisingly, recombinant Chd1 and a chimera with no leader sequence were also found in the periplasm.
authors.affiliation: Molecular Microbiology Laboratory, Department of Botany, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, PR China. jshtsang@hkucc.hku.hk
authors.forename: Jimmy S H
authors.initials: JS
authors.lastname: Tsang
authors.forename: Johnny
authors.initials: J
authors.lastname: Sze
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000502:physiology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D016956:Burkholderia cepacia / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000502:physiology*; D010957:Plasmids; D021381:Protein Transport; D011994:Recombinant Proteins / Q000096:biosynthesis; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; D000069816:SEC Translocation Channels; D006867:Hydrolases; D000251:Adenosine Triphosphatases; C034507:2-haloacid dehalogenase; D000081416:SecA Proteins