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J. Biol. Chem., Vol. 283, Issue 10, 6095-6101, March 7, 2008

This Article Full Text Full Text (PDF) All Versions of this Article: 283/10/6095    most recent M704149200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Sundaram, S. Articles by Rosen, B. P. PubMed PubMed Citation Articles by Sundaram, S. Articles by Rosen, B. P.

An Arsenate-activated Glutaredoxin from the Arsenic Hyperaccumulator Fern Pteris vittata L. Regulates Intracellular Arsenite^*

Sabarinath Sundaram, Bala Rathinasabapathi¹, Lena Q. Ma, and Barry P. Rosen^¶

From the Plant Molecular and Cellular Biology Program, Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611-0690, the Soil and Water Science Department, University of Florida, Gainesville, Florida 32611-0690, and the ^¶Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit, Michigan 48201

To elucidate the mechanisms of arsenic resistance in the arsenic hyperaccumulator fern Pteris vittata L., a cDNA for a glutaredoxin (Grx) Pv5–6 was isolated from a frond expression cDNA library based on the ability of the cDNA to increase arsenic resistance in Escherichia coli. The deduced amino acid sequence of Pv5–6 showed high homology with an Arabidopsis chloroplastic Grx and contained two CXXS putative catalytic motifs. Purified recombinant Pv5–6 exhibited glutaredoxin activity that was increased 1.6-fold by 10 mM arsenate. Site-specific mutation of Cys⁶⁷ to Ala⁶⁷ resulted in the loss of both GRX activity and arsenic resistance. PvGrx5 was expressed in E. coli mutants in which the arsenic resistance genes of the ars operon were deleted (strain AW3110), a deletion of the gene for the ArsC arsenate reductase (strain WC3110), and a strain in which the ars operon was deleted and the gene for the GlpF aquaglyceroporin was disrupted (strain OSBR1). Expression of PvGrx5 increased arsenic tolerance in strains AW3110 and WC3110, but not in OSBR1, suggesting that PvGrx5 had a role in cellular arsenic resistance independent of the ars operon genes but dependent on GlpF. AW3110 cells expressing PvGrx5 had significantly lower levels of arsenite when compared with vector controls when cultured in medium containing 2.5 mM arsenate. Our results are consistent with PvGrx5 having a role in regulating intracellular arsenite levels, by either directly or indirectly modulating the aquaglyceroporin. To our knowledge, PvGrx5 is the first plant Grx implicated in arsenic metabolism.

Received for publication, May 21, 2007 , and in revised form, December 19, 2007.

^* This work was supported by grants from the United States Department of Agriculture (Grant 2005-34135-15898) (to B. R.), the School of Natural Resources and Environment (University of Florida) (to B. R. and L. M.), and National Institutes of Health Grant R01 GM52216 (to B. P. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the Gen-Bank^TM/EBI Data Bank with accession number(s) EF052272.

¹ To whom correspondence should be addressed. Tel.: 352-392-1928, Ext. 323; Fax: 352-392-5653; E-mail: brath{at}ufl.edu.