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Mutational analysis of the Pseudomonas aeruginosa myovirus KZ morphogenetic protease gp175
Julie A Thomas1, Lindsay W Black
1Department of Biochemistry and Molecular Biology, University of Maryland Baltimore, Baltimore, Maryland, USA.
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.
Insights
The Pseudomonas aeruginosa myovirus KZ protease gp175 is a serine protease that autoactivates and cleaves inner body proteins, crucial for phage head morphogenesis. This study biochemically demonstrates gp175
Area of Science:
- Bacteriophage biology
- Structural virology
- Molecular enzymology
Background:
- Pseudomonas aeruginosa myovirus KZ possesses a unique inner body (IB) within its icosahedral capsid.
- Proteolysis by protease gp175 is essential for head and IB morphogenesis, processing numerous structural proteins.
- The precise enzymatic activity and substrate specificity of gp175 remained largely uncharacterized.
Purpose of the Study:
- To biochemically characterize the activity and substrate specificity of the purified Pseudomonas aeruginosa myovirus KZ protease gp175.
- To investigate the autoactivation mechanism and catalytic residues of gp175.
- To elucidate the cleavage patterns and N-terminal processing of IB structural proteins gp93 and gp89.
Main Methods:
- Purification and activity assays of mature gp175 and its precursor.
- Site-directed mutagenesis of predicted catalytic residues (serine, histidine) and gp93 cleavage sites.
- Analysis of proteolytic processing of IB structural proteins using purified gp175 and mutated substrates.
Main Results:
- Purified mature gp175 exhibits protease activity, cleaving IB structural proteins gp93 and gp89.
- gp175 undergoes C-terminal autoactivation, dependent on its catalytic serine and histidine residues.
- Mutations in gp93 cleavage sites blocked specific proteolysis, revealing N- to C-terminal processing and extensive N-terminal propeptide cleavage.
Conclusions:
- gp175 is a serine protease that autoactivates and plays a critical role in processing inner body structural proteins during phage morphogenesis.
- The study provides the first direct biochemical evidence for a phage protease acting on purified substrates.
- Findings have implications for understanding head morphogenesis in related giant phages, T4, and herpesviruses.
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