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Role of intermolecular disulfide bonds of the organic solvent-stable PST-01 protease in its organic solvent stability.

ABSTRACT: The PST-01 protease is secreted by the organic solvent-tolerant microorganism Pseudomonas aeruginosa PST-01 and is stable in the presence of various organic solvents. Therefore, the PST-01 strain and the PST-01 protease are very useful for fermentation and reactions in the presence of organic solvents, respectively. The organic solvent-stable PST-01 protease has two disulfide bonds (between Cys-30 and Cys-58 and between Cys-270 and Cys-297) in its molecule. Mutant PST-01 proteases in which one or both of the disulfide bonds were deleted were constructed by site-directed mutagenesis, and the effect of the disulfide bonds on the activity and the various stabilities was investigated. The disulfide bond between Cys-270 and Cys-297 in the PST-01 protease was found to be essential for its activity. The disulfide bond between Cys-30 and Cys-58 played an important role in the organic solvent stability of the PST-01 protease.

SUBMITTER: Ogino H

PROVIDER: S-EPMC92670 | biostudies-literature | 2001 Feb

REPOSITORIES: biostudies-literature

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Role of intermolecular disulfide bonds of the organic solvent-stable PST-01 protease in its organic solvent stability.

Ogino H H Uchiho T T Yokoo J J Kobayashi R R Ichise R R Ishikawa H H

Applied and environmental microbiology 20010201 2

The PST-01 protease is secreted by the organic solvent-tolerant microorganism Pseudomonas aeruginosa PST-01 and is stable in the presence of various organic solvents. Therefore, the PST-01 strain and the PST-01 protease are very useful for fermentation and reactions in the presence of organic solvents, respectively. The organic solvent-stable PST-01 protease has two disulfide bonds (between Cys-30 and Cys-58 and between Cys-270 and Cys-297) in its molecule. Mutant PST-01 proteases in which one o ...[more]

PMID: 11157266

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Project description:The GP64 envelope glycoprotein of the Autographa californica nucleopolyhedrovirus (AcMNPV) is a class III viral membrane fusion protein that is triggered by low pH during entry. Unlike most other viral fusion protein trimers, the monomers of GP64 are covalently linked to each other within the trimer by a single intermolecular disulfide bond (Cys24 Cys372). Single or paired alanine substitutions for Cys24 and Cys372 resulted in lower-efficiency transport of GP64 to the cell surface. Surprisingly, these mutated GP64s induced syncytium formation, and normalized fusion activities were approximately 30% of that from wild-type (WT) GP64. Heat treatment (37 degrees C) did not further reduce fusion activity of GP64 constructs with a disrupted intermolecular disulfide bond, suggesting that the GP64 trimers were relatively thermostable in the absence of the intermolecular disulfide bond. In addition, analysis of binding by a conformation-specific monoclonal antibody (MAb) suggested that the low-pH-induced refolding of those GP64 constructs was generally similar to that of WT GP64. In addition to its critical role in membrane fusion, GP64 is also necessary for efficient budding. When GP64 constructs containing a disrupted intermolecular disulfide bond (Cys24 Cys372) were displayed at the cell surface at levels comparable to those of WT GP64, virion budding efficiency ranged from approximately 39 to 88%, indicating that the intermolecular disulfide bond is not required for virion budding. However, GP64 proteins with a disrupted intermolecular disulfide could not rescue a GP64-null bacmid. We also examined the 6 conserved intramolecular disulfide bonds using single and paired alanine substitution mutations. None of the GP64 constructs with disrupted intramolecular disulfide bonds were capable of mediating pH-triggered membrane fusion, indicating that the intramolecular disulfide bonds are all necessary for membrane fusion. Thus, while the intramolecular disulfide bonds of GP64 appear to serve critical roles in membrane fusion, the unusual intermolecular disulfide bond was not critical for membrane fusion or virion budding yet appears to play an unknown role in viral infectivity.

Dataset Information

Role of intermolecular disulfide bonds of the organic solvent-stable PST-01 protease in its organic solvent stability.

Publications

Role of intermolecular disulfide bonds of the organic solvent-stable PST-01 protease in its organic solvent stability.

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