Engineering of 2-Cys Peroxiredoxin for En-hanced Stress-Tolerance

다국어 초록 (Multilingual Abstract)

A typical 2-cysteine peroxiredoxin (2-Cys Prx)-like protein (PpPrx) that alternatively acts as a peroxidase or a mole-cular chaperone in Pseudomonas putida KT2440 was previously characterized. The dual functions of PpPrx are regulated by the existence of an additional Cys^112 between the active Cys^51 and Cys^171 residues. In the present study, additional Cys residues (Cys^31, Cys^112, and Cys^192) were added to PpPrx variants to improve their enzymatic func-tion. The optimal position of the additional Cys residues for the dual functionality was assessed. The peroxidase activities of the S31C and Y192C mutants were increased 3- to 4-fold compared to the wild-type, while the chaperone activity was maintained at > 66% of PpPrx. To investigate whether optimization of the dual functions could enhance stress-tolerance in vivo, a complementation study was performed. The S31C and Y192C mutants showed a much greater tolerance than other variants under a complex condition of heat and oxidative stresses. The optimized dual functions of PpPrx could be adapted for use in bioen-gineering systems and industries, such as to develop or-ganisms that are more resistant to extreme environments.

참고문헌 (Reference)

1 Jang, H.H., "Two enzymes in one; two yeast peroxiredoxins display oxidative stress-dependent switching from a peroxidase to a molecular chaperone function" 117 : 625-635, 2004

2 Gnanasekar, M., "Translationally controlled tumor protein is a novel heat shock protein with chaperone-like activity" 386 : 333-337, 2009

3 Chuang, M.H., "The antioxidant protein alkyl hydroperoxide reductase of helicobacter pylori switches from a peroxide reductase to a molecular chaperone function" 103 : 2552-2557, 2006

4 Wood, Z.A., "Structure, mechanism and regulation of peroxiredoxins" 28 : 32-40, 2003

5 Jang, H.H., "Structural and functional regulation of eukaryotic 2-Cys peroxiredoxins including the plant ones in cellular defense-signaling mechanisms against oxidative stress" BLACKWELL PUBLISHING 126 : 549-559, 2006

6 Chang, T.-S., "Regulation of peroxiredoxin I activity by Cdc2-mediated phosphorylation" 277 : 25370-25376, 2002

7 Cumming, R.C., "Protein disulfide bond formation in the cytoplasm during oxidative stress" 279 : 21749-21758, 2004

8 Shi, L., "Protein conformational changes induced by 1,1′-bis(4-anilino-5-naphthalenesulfonic acid):preferential binding to the molten globule of DnaK" 33 : 7536-7546, 1994

9 Kristensen, P., "Properties of thiol-specific anti-oxidant protein or calpromotin in solution" 262 : 127-131, 1999

10 Schröder, E., "Porcine naturalkiller-enhancing factor-B: oligomerisation and identification as a calpain substrate in vitro" 1383 : 279-291, 1998

11 Dietz, K.J., "Plant peroxiredoxins" 54 : 93-107, 2003

12 Ito, H., "Phosphorylation-induced change of the oligomerization state of alpha B-crystallin" 276 : 5346-5352, 2001

13 Jang, H.H., "Phosphorylation and concomitant structural changes in human 2-Cys peroxiredoxin isotype I differentially regulate its peroxidase and molecular chaperone functions" ELSEVIER SCIENCE BV 580 : 351-355, 2006

14 Hofmann, B., "Peroxiredoxins" 383 : 347-364, 2002

15 Moon, J.C., "Oxidative stress-dependent" American Society for Biochemistry and Molecular Biology Inc. 280 (280): 28775-28784, 2005

16 Cheong, N.E., "Molecular cloning, expression, and functional characterization of a 2 Cys-peroxiredoxin in Chinese cabbage" 40 : 825-834, 1999

17 Sharma, K.K., "Interaction of 1,1′-bi(4-anilino)naphthalene-5,5′-disulfonic acid with alpha-crystallin" 273 : 8965-8970, 1998

18 우현애, "Inactivation of human peroxiredoxin I during catalysis as the result of the oxidation of the catalytic site cysteine to cysteine-sulfinic acid" 277 (277): 38029-38036, 200210

19 An, B.C., "Functional switching of a novel prokaryotic 2-Cys peroxiredoxin (PpPrx) under oxidative stress" 16 : 317-328, 2011

20 Link, A.J., "Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli K-12" 18 : 1259-1313, 1997

21 Chae, H.Z., "Cloning and sequencing of thiol-specific antioxidant from mammalian brain: alkyl hydroperoxide reductase and thiol-specific antioxidant define a large family of antioxidant enzymes" 91 : 7017-7021, 1994

22 Chae, H.Z., "Characterization of three isoforms of mammalian peroxiredoxin that reduce peroxides in the presence of thioredoxin" 45 : 101-112, 1999

23 Ganea, E., "Chaperone-like activity of alpha-crystallin and other small heat shock proteins" 2 : 205-225, 2001

24 Baier, M., "Alkyl hydroperoxide reductase: the way out of the oxidative breakdown of lipids in chloroplast" 4 : 166-168, 1999

25 Lee, G.J., "A small heat shock protein stably binds heat-denatured model substrates and can maintain a substrate in a folding-competent state" 16 : 659-671, 1997

26 Bradford, M.M., "A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding" 72 : 248-254, 1976

27 Jeong, W.J., "A new member of human Tsa/AhpC as thioredoxin-dependent thiol peroxidase" 33 : 234-241, 2000

28 Byung Chull An, "A New Antioxidant with Dual Functions as a Peroxidase and Chaperone in Pseudomonas aeruginosa" 한국분자세포생물학회 29 (29): 145-151, 2010