title: Structural-functional characterization and physiological significance of ferredoxin-NADP reductase from Xanthomonas axonopodis pv. citri.
abstract: Xanthomonas axonopodis pv. citri is a phytopathogen bacterium that causes severe citrus canker disease. Similar to other phytopathogens, after infection by this bacterium, plants trigger a defense mechanism that produces reactive oxygen species. Ferredoxin-NADP(+) reductases (FNRs) are redox flavoenzymes that participate in several metabolic functions, including the response to reactive oxygen species. Xanthomonas axonopodis pv. citri has a gene (fpr) that encodes for a FNR (Xac-FNR) that belongs to the subclass I bacterial FNRs. The aim of this work was to search for the physiological role of this enzyme and to characterize its structural and functional properties. The functionality of Xac-FNR was tested by cross-complementation of a FNR knockout Escherichia coli strain, which exhibit high susceptibility to agents that produce an abnormal accumulation of (•)O(2)(-). Xac-FNR was able to substitute for the FNR in E. coli in its antioxidant role. The expression of fpr in X. axonopodis pv. citri was assessed using semiquantitative RT-PCR and Western blot analysis. A 2.2-fold induction was observed in the presence of the superoxide-generating agents methyl viologen and 2,3-dimethoxy-1,4-naphthoquinone. Structural and functional studies showed that Xac-FNR displayed different functional features from other subclass I bacterial FNRs. Our analyses suggest that these differences may be due to the unusual carboxy-terminal region. We propose a further classification of subclass I bacterial FNRs, which is useful to determine the nature of their ferredoxin redox partners. Using sequence analysis, we identified a ferredoxin (XAC1762) as a potential substrate of Xac-FNR. The purified ferredoxin protein displayed the typical broad UV-visible spectrum of [4Fe-4S] clusters and was able to function as substrate of Xac-FNR in the cytochrome c reductase activity. Our results suggest that Xac-FNR is involved in the oxidative stress response of Xanthomonas axonopodis pv. citri and performs its biological function most likely through the interaction with ferredoxin XAC1762.
authors.affiliation: Molecular Biology Division, Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.
authors.forename: María Laura
authors.initials: ML
authors.lastname: Tondo
authors.forename: Matías A
authors.initials: MA
authors.lastname: Musumeci
authors.forename: María Laura
authors.initials: ML
authors.lastname: Delprato
authors.forename: Eduardo A
authors.initials: EA
authors.lastname: Ceccarelli
authors.forename: Elena G
authors.initials: EG
authors.lastname: Orellano
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D017433:Protein Structure, Secondary; D053532:Xanthomonas axonopodis / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D005287:Ferredoxin-NADP Reductase
references.citation: Biochemistry. 2005 Sep 6;44(35):11730-40
references.pmid: 16128574
references.citation: Plant Mol Biol. 1994 Oct;26(2):679-90
references.pmid: 7948922
references.citation: BMC Struct Biol. 2007 Oct 24;7:69
references.pmid: 17958910
references.citation: Anal Biochem. 1977 May 1;79(1-2):544-52
references.pmid: 68686
references.citation: J Biol Chem. 2004 Oct 22;279(43):44590-9
references.pmid: 15308657
references.citation: Eur J Biochem. 2003 May;270(9):1900-15
references.pmid: 12709048
references.citation: PLoS One. 2010 May 24;5(5):e10803
references.pmid: 20520822
references.citation: J Bacteriol. 1995 Aug;177(15):4528-31
references.pmid: 7635836
references.citation: FEBS Lett. 1995 Jul 17;368(2):220-4
references.pmid: 7628609
references.citation: J Bacteriol. 2009 Mar;191(5):1472-9
references.pmid: 19114475
references.citation: J Bacteriol. 2001 Jul;183(13):3890-902
references.pmid: 11395452
references.citation: Plant Physiol. 2000 Jul;123(3):1037-45
references.pmid: 10889253
references.citation: Arch Biochem Biophys. 1956 Dec;65(2):475-90
references.pmid: 13395503
references.citation: J Bacteriol. 2002 Mar;184(5):1474-80
references.pmid: 11844783
references.citation: Biochemistry. 2011 Mar 29;50(12):2111-22
references.pmid: 21306142
references.citation: Biochemistry. 1992 Aug 11;31(31):7061-8
references.pmid: 1643039
references.citation: Biochim Biophys Acta. 1976 Aug 12;445(1):14-24
references.pmid: 8135
references.citation: J Mol Biol. 1996 Jul 19;260(3):289-98
references.pmid: 8757792
references.citation: Biochemistry. 2007 Oct 30;46(43):12198-211
references.pmid: 17915950
references.citation: Biochem J. 1997 Mar 15;322 ( Pt 3):681-92
references.pmid: 9148737
references.citation: J Biol Chem. 1999 Jan 29;274(5):2978-87
references.pmid: 9915836
references.citation: Biochemistry. 2006 Nov 21;45(46):13899-909
references.pmid: 17105208
references.citation: Biochemistry. 1995 Oct 3;34(39):12842-8
references.pmid: 7548039
references.citation: Biochemistry. 1992 Sep 15;31(36):8473-81
references.pmid: 1390631
references.citation: J Biol Chem. 1993 Sep 15;268(26):19267-73
references.pmid: 8366077
references.citation: Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1328-31
references.pmid: 8108411
references.citation: J Bacteriol. 1999 Aug;181(15):4639-43
references.pmid: 10419964
references.citation: Microbiology (Reading). 2006 Apr;152(Pt 4):1119-1128
references.pmid: 16549675
references.citation: Biochem Biophys Res Commun. 2006 Jan 27;339(4):1246-54
references.pmid: 16360643
references.citation: FASEB J. 1997 Feb;11(2):133-40
references.pmid: 9039955
references.citation: J Biol Chem. 1965 Mar;240:1405-11
references.pmid: 14284756
references.citation: Photosynth Res. 2004 Feb;79(2):113-31
references.pmid: 16228387
references.citation: J Biochem. 1999 Jul;126(1):10-8
references.pmid: 10393315
references.citation: Proc Natl Acad Sci U S A. 1990 Aug;87(16):6181-5
references.pmid: 1696718
references.citation: Biochim Biophys Acta. 2004 May 6;1698(2):155-65
references.pmid: 15134648
references.citation: J Mol Biol. 2007 Mar 23;367(2):501-13
references.pmid: 17258767
references.citation: Biochemistry. 1971 Jan 5;10(1):113-24
references.pmid: 5099590
references.citation: J Biol Chem. 1994 Jul 29;269(30):19404-9
references.pmid: 8034707
references.citation: Nature. 2002 May 23;417(6887):459-63
references.pmid: 12024217
references.citation: Mol Plant Pathol. 2003 May 1;4(3):141-57
references.pmid: 20569374
references.citation: Biochim Biophys Acta. 2009 Feb;1794(2):199-210
references.pmid: 18973834
references.citation: Biochemistry. 2009 Jun 23;48(24):5760-8
references.pmid: 19435322
references.citation: Eur J Biochem. 1997 Mar 1;244(2):361-70
references.pmid: 9119001
references.citation: Nat Rev Microbiol. 2004 Dec;2(12):954-66
references.pmid: 15550941
references.citation: Biochem Biophys Res Commun. 2006 Mar 3;341(1):51-6
references.pmid: 16412384
references.citation: J Biol Chem. 2000 Apr 7;275(14):10472-6
references.pmid: 10744737
references.citation: J Bacteriol. 2009 Dec;191(23):7372-7
references.pmid: 19783631
references.citation: J Bacteriol. 1993 Mar;175(6):1590-5
references.pmid: 8449868
references.citation: Methods Enzymol. 1987;153:3-11
references.pmid: 3323803
references.citation: FEBS Lett. 2003 Oct 23;553(3):408-12
references.pmid: 14572660
references.citation: Mol Plant Pathol. 2004 Jan 1;5(1):1-15
references.pmid: 20565577
references.citation: J Bacteriol. 2000 Dec;182(23):6842-4
references.pmid: 11073934