Isolation and characterization of new C-terminal substitution mutations affecting secretion of polygalacturonase in Erwinia carotovora ssp. carotovora.
An intact C-terminus was previously shown to be required for stability and secretion of the polygalacturonase (PehA) in Erwinia carotovora ssp. carotovora. Here we have analyzed the effects of amino acid (aa) substitutions generated to five C-terminal positions of PehA. Conservation of two hydrophobic and one non-hydrophobic residue (V372, V374 and N371, respectively) was found to be essential for maintenance of the protein stability. As an exception, one of the mutants (V372G) did not show major effects on protein stability, as determined by immunoblots and enzyme activity assay, yet it prevented the secretion completely. We conclude that the C-terminus of PehA is directly involved in the formation or stabilization of a conformation-sensitive structure needed for recognition of the protein as secreted.
Palomäki
T
T
Department of Biosciences, Division of Genetics, Biocenter 2, University of Helsinki, Finland.
Saarilahti
H T
HT
D001665:Binding Sites; D001692:Biological Transport; D004795:Enzyme Stability; D004926:Escherichia coli; D016973:Pectobacterium carotovorum / Q000201:enzymology* / Q000235:genetics; D017354:Point Mutation; D011096:Polygalacturonase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics
D011994:Recombinant Proteins; D011096:Polygalacturonase