Biosynthesis and secretion of pullulanase, a lipoprotein from Klebsiella aerogenes.
We constructed, by site-directed mutagenesis, a mutant pullulanase gene in which the cysteine residue in a pentapeptide sequence, Leu16-Leu-Ser-Gly-Cys20 within the NH2-terminal region of pullulanase from Klebsiella aerogenes, is replaced by serine (Ser20). The modification, processing, and subcellular localization of the mutant pullulanase were studied. Labeling studies with [3H]palmitate and immunoprecipitation with mouse antiserum raised against pullulanase showed that the wild form of both the extracellular and intracellular pullulanases contained lipids, whereas the mutant enzyme was not modified with lipids. Only the Cys20 was modified with glyceryl lipids. The bulk of the mutant pullulanase was located in the periplasm, but a portion of the unmodified, mutant pullulanase was secreted into the medium. Mutant pullulanases from the extracellular and the periplasm were purified and their NH2-terminal sequences were determined. Both the mutant pullulanases were cleaved between residues of Ser13 and Leu14 which is 6-amino acid residues upstream of the lipid modified pullulanase cleavage site. This new cleavage was resistant to globomycin, an inhibitor of the prolipoprotein signal peptidase of Escherichia coli. These results indicate that the pentapeptide sequence plays an important role in maturation and translocation of pullulanase in K. aerogenes. However, the modification of pullulanase with lipids seems to be not essential for export of the enzyme across the outer membrane.
Murooka
Y
Y
Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Japan.
Ikeda
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism; D007163:Immunosorbent Techniques; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism
D010169:Palmitic Acids; D010446:Peptide Fragments; D011498:Protein Precursors; D019308:Palmitic Acid; D006026:Glycoside Hydrolases; C100181:pullulanase