title: Redox properties of a thioredoxin-like Arabidopsis protein, AtTDX.
abstract: AtTDX is an enzyme present in Arabidopsis thaliana which is composed of two domains, a thioredoxin (Trx)-motif containing domain and a tetratricopeptide (TPR)-repeat domain. This enzyme has been shown to function as both a thioredoxin and a chaperone. The midpoint potential (E(m)) of AtTDX was determined by redox titrations using the thiol-specific modifiers, monobromobimane (mBBr) and mal-PEG. A NADPH/Trx reductase (NTR) system was used both to validate these E(m) determination methods and to demonstrate that AtTDX is an electron-accepting substrate for NTR. Titrations of full-length AtTDX revealed the presence of a single two-electron couple with an E(m) value of approximately -260 mV at pH 7.0. The two cysteines present in a typical, conserved Trx active site (WCGPC), which are likely to play a role in the electron transfer processes catalyzed by AtTDX, have been replaced by serines by site-directed mutagenesis. These replacements (i.e., C304S, C307S, and C304S/C307S) resulted in a complete loss of the redox process detected using either the mBBr or mal-PEG method to monitor disulfide/dithiol redox couples. This result supports the conclusion that the couple with an E(m) value of -260 mV is a disulfide/dithiol couple involving Cys304 and Cys307. Redox titrations for the separately-expressed Trx-motif containing C-domain also revealed the presence of a single two-electron couple with an E(m) value of approximately -260 mV at 20°C. The fact that these two E(m) values are identical, provides additional support for assignment of the redox couple to a disulfide/dithiol involving C304 and C307. It was found that, while the disulfide/dithiol redox chemistry of AtTDX was not affected by increasing the temperature to 40°C, no redox transitions were observed at 50°C and higher temperatures. In contrast, Escherichia coli thioredoxin was shown to remain redox-active at temperatures as high as 60°C. The temperature-dependence of the AtTDX redox titration is similar to that observed for the redox activity of the protein in enzymatic assays.
authors.affiliation: Department of Chemistry and Biochemistry, Baylor University, Waco, TX 76798-7348, USA; Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701, Republic of Korea.
authors.forename: Sang Gon
authors.initials: SG
authors.lastname: Kim
authors.forename: Yong Hun
authors.initials: YH
authors.lastname: Chi
authors.forename: Jong-Sun
authors.initials: JS
authors.lastname: Lee
authors.forename: Sara Rae
authors.initials: SR
authors.lastname: Schlesinger
authors.forename: Masoud
authors.initials: M
authors.lastname: Zabet-Moghaddam
authors.forename: Jung-Sung
authors.initials: JS
authors.lastname: Chung
authors.forename: David B
authors.initials: DB
authors.lastname: Knaff
authors.forename: Sun Tae
authors.initials: ST
authors.lastname: Kim
authors.forename: Sang Yeol
authors.initials: SY
authors.lastname: Lee
authors.forename: Sung-Kun
authors.initials: SK
authors.lastname: Kim
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014050:Toluene / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; C504764:AtTDX protein, Arabidopsis; D004220:Disulfides; D014050:Toluene; D012694:Serine; D013879:Thioredoxins; D013880:Thioredoxin-Disulfide Reductase; D003545:Cysteine; C004848:dithiol