title: Substitution of Thr(55) by Gly and Lys(48) by Asp in OsTrx20 using site-directed mutagenesis.
abstract: Thioredoxins are small (12-13kDa) ubiquitous proteins containing a redox active disulfide bridge. The primary structure of one of the rice Trx isoforms, OsTrx20, in which Thr is substituted for the largely conserved Gly in position 55 in the active site and Lys is substituted for the conserved Asp/Asn in position 48 is considerably different with other h-type Trx isoforms. In order to probe the functional roles of Thr-55 and Lys-48 in OsTrx20, Thr was replaced with Gly and Lys with Asp using site-directed mutagenesis. The wild type OsTrx20 as well as single mutants T55GOsTrx20, K48DOsTrx20 and the double mutant T55G-K48DOstrx20 were heterologously expressed in Escherichia coli and purified. The changes in the ability to reduce insulin for OsTrx20 and mutants as well as OsTrx23 which has a Trx typical active site were monitored in the pH range 6.5-8. The results showed that whereas the activity of wild type OsTrx20 is dependent on pH and decreases remarkably at high pH values, the activities of mutants T55GOsTrx20, K48DOsTrx20, T55G-K48DOsTrx20 and wild type OsTrx23 slightly change under different pH conditions. These results support the significant involvement of residues Thr-55 and Lys-48 in instability of OsTrx20 activity under pH variations.
authors.affiliation: Department of Agricultural Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan 84156-83111, Iran.
authors.forename: Mitra
authors.initials: M
authors.lastname: Roodgar Nashta
authors.affiliation: Department of Agricultural Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan 84156-83111, Iran. Electronic address: a.shahpiri@cc.iut.ac.ir.
authors.forename: Azar
authors.initials: A
authors.lastname: Shahpiri
authors.affiliation: Department of Biomedical Engineering, College of Engineering, University of Isfahan, Isfahan 81746-73441, Iran.
authors.forename: Majid
authors.initials: M
authors.lastname: Mohammad Beigi
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D000596:Amino Acids / Q000235:genetics*; D055162:Biocatalysis; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics; D012275:Oryza / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics*; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D012995:Solubility; D013879:Thioredoxins / Q000235:genetics*; D014170:Transformation, Genetic
chemical_list: D000596:Amino Acids; D007328:Insulin; D050505:Mutant Proteins; D010940:Plant Proteins; D013879:Thioredoxins
keywords: OsTrx20; Rice; Site-directed mutagenesis; Thioredoxin