[국내논문]Enhancement of the Chaperone Activity of Alkyl Hydroperoxide Reductase C from Pseudomonas aeruginosa PAO1 Resulting from a Point-Specific Mutation Confers Heat Tolerance in Escherichia coli원문보기
Alkyl hydroperoxide reductase subunit C from Pseudomonas aeruginosa PAO1 (PaAhpC) is a member of the 2-Cys peroxiredoxin family. Here, we examined the peroxidase and molecular chaperone functions of PaAhpC using a site-directed mutagenesis approach by substitution of Ser and Thr residues with Cys at...
Alkyl hydroperoxide reductase subunit C from Pseudomonas aeruginosa PAO1 (PaAhpC) is a member of the 2-Cys peroxiredoxin family. Here, we examined the peroxidase and molecular chaperone functions of PaAhpC using a site-directed mutagenesis approach by substitution of Ser and Thr residues with Cys at positions 78 and 105 located between two catalytic cysteines. Substitution of Ser with Cys at position 78 enhanced the chaperone activity of the mutant (S78C-PaAhpC) by approximately 9-fold compared with that of the wild-type protein (WT-PaAhpC). This increased activity may have been associated with the proportionate increase in the high-molecular-weight (HMW) fraction and enhanced hydrophobicity of S78C-PaAhpC. Homology modeling revealed that mutation of $Ser^{78}$ to $Cys^{78}$ resulted in a more compact decameric structure than that observed in WT-PaAhpC and decreased the atomic distance between the two neighboring sulfur atoms of $Cys^{78}$ in the dimer-dimer interface of S78C-PaAhpC, which could be responsible for the enhanced hydrophobic interaction at the dimer-dimer interface. Furthermore, complementation assays showed that S78C-PaAhpC exhibited greatly improved the heat tolerance, resulting in enhanced1 survival under thermal stress. Thus, addition of Cys at position 78 in PaAhpC modulated the functional shifting of this protein from a peroxidase to a chaperone.
Alkyl hydroperoxide reductase subunit C from Pseudomonas aeruginosa PAO1 (PaAhpC) is a member of the 2-Cys peroxiredoxin family. Here, we examined the peroxidase and molecular chaperone functions of PaAhpC using a site-directed mutagenesis approach by substitution of Ser and Thr residues with Cys at positions 78 and 105 located between two catalytic cysteines. Substitution of Ser with Cys at position 78 enhanced the chaperone activity of the mutant (S78C-PaAhpC) by approximately 9-fold compared with that of the wild-type protein (WT-PaAhpC). This increased activity may have been associated with the proportionate increase in the high-molecular-weight (HMW) fraction and enhanced hydrophobicity of S78C-PaAhpC. Homology modeling revealed that mutation of $Ser^{78}$ to $Cys^{78}$ resulted in a more compact decameric structure than that observed in WT-PaAhpC and decreased the atomic distance between the two neighboring sulfur atoms of $Cys^{78}$ in the dimer-dimer interface of S78C-PaAhpC, which could be responsible for the enhanced hydrophobic interaction at the dimer-dimer interface. Furthermore, complementation assays showed that S78C-PaAhpC exhibited greatly improved the heat tolerance, resulting in enhanced1 survival under thermal stress. Thus, addition of Cys at position 78 in PaAhpC modulated the functional shifting of this protein from a peroxidase to a chaperone.
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문제 정의
In this study, we aimed to determine the role of protein structure and amino acid sequence in mediating the dual functions of PaAhpC. Our results provided important insights into the functional switching of PaAhpC between its peroxidase and chaperone functions and the capacity for the mutant proteins to support the bacterial cell to cope up with abiotic stresses.
가설 설정
, 2007). Therefore, we hypothesize that the reduced distance between Cys78 residues in the dimer-dimer interface of S78C-PaAhpC enhances the hydrophobic interactions and/or van der Waals forces and facilitates the conversion of PaAhpC from the LMW structure to the HMW structure, which exhibits increased chaperone function.
In this study, we demonstrated that PaAhpC exhibited dual functionalities as a peroxidase and a molecular chaperone. However, as a member of the 2-Cys Prx family of proteins and due to its intrinsic antioxidant properties, WT-PaAhpC preferred to act as a peroxidase rather than a molecular chaperone.
제안 방법
Therefore, in the current study, we aimed to determine the roles of these two additional cysteines in modulating the dual functions of PaAhpC. For this purpose, two mutant PaAhpC proteins were generated by substituting Ser and Thr at positions 78 and 105 with Cys through site-directed mutagenesis and then evaluated the ability of the mutated PaAhpC to confer resistance to oxidative and heat stress in complemented E. coli. The structure and function of 2-Cys Prx of Pseudomonas species have already been well characterized and standardized in our laboratory (An et al.
The protein sequence of PaAhpC was set as the query in the protein data bank, and PSI-BLAST was selected as the algorithm for the BLAST search. The sequence alignment was performed using Clustal W2 web-based software in EMBL-EBI (www.
jp) of National Institute of Genetics. To test the expression levels of PaAhpC protein, we examined the immunoblot assay probed with a polyclonal PaAhpC antibodies using the transformed E. coli or P. aerusinosa with optical density (OD) of 0.9-1.0 at 600 nm.
Condition-dependent conformational changes from a LMW structure to HMW structures triggered the shift in PaAhpC function from a peroxidase to a chaperone. Furthermore, the ability of PaAhpC to perform chaperone activity was greatly improved by substituting Cys for Ser at position 78 (S78C-PaAhpC), located between the two conserved catalytic cysteines, using site-directed mutagenesis. S78C-PaAhpC, which exhibited enhanced chaperone activity, had markedly increased heat tolerance.
이론/모형
uk). The Generate Homology Modeling protocol in Discovery Studio (DS) 3.5 (Accelrys Software Inc.; Accelrys, USA) was used for homology modeling of PaAhpC. After homology modeling, the stereochemical qualities of the homology models were validated using PROCHECK (Laskowski et al.
성능/효과
brasilense. Our results provided important insights into the functional shifting of PaAhpC between its peroxidase and chaperone functions and the capacity for the mutant proteins to deal with abiotic stresses.
, 2012). Additionally, native PAGE and SEC analyses also showed that the HMW structure was more common in the S78C-PaAhpC mutant than in WT-PaAhpC. The HMW fraction from SEC corresponded to low peroxidase activity and high chaperone activity.
S78C-PaAhpC, which exhibited enhanced chaperone activity, had markedly increased heat tolerance. Thus, our results indicated that the addition of a Cys residue at position 78 in PaAhpC modulated the functional shifting of this protein from a peroxidase to a chaperone. This feature may be exploited in the further development of heat-tolerant bacteria for industrial applications.
후속연구
This feature may be exploited in the further development of heat-tolerant bacteria for industrial applications. Additional studies are required to further elucidate the mechanisms modulating the dual functions of PaAhpC and other 2-Cys Prx proteins to improve our understanding of the functional shifting from a peroxidase to a chaperone and enhancement of abiotic stress tolerance.
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