title: Mutational analysis of the Pseudomonas aeruginosa myovirus KZ morphogenetic protease gp175.
abstract: Pseudomonas aeruginosa myovirus KZ has a 270-kb genome within a T=27 icosahedral capsid that contains a large, unusual, and structurally well-defined protein cylindrical inner body (IB) spanning its interior. Proteolysis forms a pivotal stage in KZ head and IB morphogenesis, with the protease gp175 cleaving at least 19 of 49 different head proteins, including the major capsid protein and five major structural IB proteins. Here we show that the purified mature form of gp175 is active and cleaves purified IB structural proteins gp93 and gp89. Expression vector synthesis and purification of the zymogen/precursor yielded an active, mature-length protease, showing independent C-terminal gp175 self-cleavage autoactivation. Mutation of either the predicted catalytic serine or histidine inactivated mature gp175, supporting its classification as a serine protease and representing the first such direct biochemical demonstration with purified protease and substrate proteins for any phage protease. These mutations also blocked self-cleavage of the precursor while allowing intermolecular gp175 processing. To confirm the cleavage specificity of gp175, we mutated three cleavage sites in gp93, which blocked proteolysis at these sites. The N-terminal propeptide of gp93 was shown to undergo more extensive proteolysis than previously identified. We found that proteolysis in gp93 progressed from the N to C terminus, while blocking cleavage sites slowed but did not eliminate downstream proteolysis. These findings were shown by informatics to be relevant to the head morphogenesis of numbers of other related IB-containing giant phages as well as to T4 and herpesviruses, which have homologous proteases.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland Baltimore, Baltimore, Maryland, USA.
authors.forename: Julie A
authors.initials: JA
authors.lastname: Thomas
authors.forename: Lindsay W
authors.initials: LW
authors.lastname: Black
mesh_terms: D004252:DNA Mutational Analysis; D050505:Mutant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017900:Myoviridae / Q000201:enzymology* / Q000235:genetics; D017105:Pseudomonas Phages / Q000201:enzymology* / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000821:virology; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D057057:Serine Proteases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D057057:Serine Proteases