In vitro analysis of roles of a disulfide bridge and a calcium binding site in activation of Pseudomonas sp. strain KWI-56 lipase.
The expression of lipase from Pseudomonas sp. strain KWI-56 (recently reclassified as Burkholderia cepacia) had been found to be dependent on an activator gene (act) downstream of its structural gene (lip). In this work, the mature lipase was synthesized in an enzymatically active form with a cell-free Escherichia coli S30 coupled transcription-translation system by expressing a recombinant lipase gene (rlip) encoding the mature lipase in the presence of its purified activator or by coexpression of rlip and act. The in vitro expression systems were used for studying the folding process of the lipase. The addition of dithiothreitol in the expression systems decreased the activity dramatically without affecting the synthesis level of the lipase, whereas the in vitro-synthesized active lipase was relatively stable even in the presence of dithiothreitol. This phenomenon was further investigated by constructing mutant lipase genes only in vitro by PCR without gene cloning. Replacements of cysteine residues (Cys190 and Cys270) forming a sole putative disulfide bond to serine residues decreased the lipase activity greatly, suggesting that the disulfide bond was essential for the proper folding of the lipase. In addition, replacing Asp242 and Asp288, which were deduced to be part of a Ca(2+) binding site, also greatly decreased the activities of the in vitro-synthesized lipases. The role of the Ca(2+) binding site in the activation of the lipase is also discussed.
Yang
J
J
Laboratory of Molecular Biotechnology, Graduate School of Biological and Agricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan.
Kobayashi
K
K
Iwasaki
Y
Y
Nakano
H
H
Yamane
T
T
D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011549:Pseudomonas / Q000187:drug effects / Q000201:enzymology*
D004220:Disulfides; D008049:Lipase; D002118:Calcium; D004229:Dithiothreitol