Expression, mutagenesis and kinetic analysis of recombinant K1E endosialidase to define the site of proteolytic processing and requirements for catalysis.
Catalytically active, recombinant fusion proteins of bacteriophage E endosialidase were expressed and purified from Escherichia coli. Constructs with different fusion partners added to the amino terminus of the endosialidase were enzymatically active. A post-translational proteolytic cleavage was shown to occur between serine 706 and aspartate 707 to generate the 76 kDa mature enzyme from the 90 kDa translation product. Endosialidase truncated at the C-terminus from aspartate 707 was observed to have the same 76 kDa molecular weight as wild-type enzyme using denaturing SDS-PAGE but, under native PAGE conditions, was not observed to form the approximately 250 kDa trimeric wild-type enzyme, implying that the C-terminus of the enzyme may be required for correct assembly of active trimer, rather than as part of the active site as has been previously suggested. Mutagenesis of aspartate 138 to alanine greatly reduced enzyme activity whereas conversion of other selected aspartate residues to alanine had less effect, consistent with similarities between the structure and cata-lytic mechanism of bacteriophage E endosialidase and those of exosialidases.
Leggate
Daniel R
DR
Department of Clinical Biochemistry and Cambridge Institute for Medical Research, University of Cambridge, Addenbrooke's Hospital, UK.
Bryant
J Mark
JM
Redpath
Maria B
MB
Head
Denise
D
Taylor
Peter W
PW
Luzio
J Paul
JP
D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001435:Bacteriophages / Q000201:enzymology*; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009439:Neuraminidase / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D001224:Aspartic Acid; C035355:endo-N-acetylneuraminidase; D009439:Neuraminidase; D000409:Alanine