Molecular characterization of pulA and its product, pullulanase, a secreted enzyme of Klebsiella pneumoniae UNF5023.
The determined nucleotide sequence of the Klebsiella pneumoniae UNF5023 gene pulA comprises a single open reading frame coding for a 1090-residue precursor of the secreted protein pullulanase. The predicted sequence of this protein is highly homologous to that of pullulanase of Klebsiella aerogenes strain W70. However, the UNF5023 pullulanase lacks a collagen-like sequence present at the N-terminus of the mature W70 enzyme and differs further from the W70 pullulanase around residue 300 and at the C-terminus. Pullulanases with or without the collagen-like sequence could not be separated by gel electrophoresis under denaturing or non-denaturing conditions, and were unaffected by collagenase. A large central domain which is highly conserved in both UNF5023 and W70 polypeptides contains eight short sequences that are also found in amylases and iso-amylases. Linker mutations in the region of the UNF5023 pulA gene coding for this domain abolished catalytic activity without affecting transport of the polypeptide across the outer membrane. Hybrid proteins comprising at least the amino-terminal 656 residues of prepullulanase fused to alkaline phosphatase were partially localized to the cell surface, as judged by their accessibility to anti-pullulanase serum in immuno-fluorescence tests. On the basis of these results, we tentatively propose that secretion signals required for recognition and translocation across the outer membrane via the pullulanase-specific extension of the secretion pathway are located near the N-terminus of the pullulanase polypeptide.
Kornacker
M G
MG
Unité de Génétique Moléculaire, Institut Pasteur, Paris, France.
Pugsley
A P
AP
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003094:Collagen; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000235:genetics*; D007711:Klebsiella pneumoniae / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D015183:Restriction Mapping
D003094:Collagen; D006026:Glycoside Hydrolases; C100181:pullulanase