Substituting selenocysteine for catalytic cysteine 41 enhances enzymatic activity of plant phospholipid hydroperoxide glutathione peroxidase expressed in Escherichia coli.
The citrus phospholipid hydroperoxide glutathione peroxidase (cit-PHGPx) was the first plant peroxidase demonstrated to exhibit PHGPx-specific enzymatic activity, although it was 500-fold weaker than that of the pig heart analog. This relatively low activity is accounted for the catalytic residue of cit-PHGPx, which was found to be cysteine and not the rare selenocysteine (Sec) present in animal enzymes. Sec incorporation into proteins is encoded by a UGA codon, usually a STOP codon, which, in prokaryotes, is suppressed by an adjacent downstream mRNA stem-loop structure, the Sec insertion sequence (SECIS). By performing appropriate nucleotide substitutions into the gene encoding cit-PHGPx, we introduced bacterial-type SECIS elements that afforded the substitution of the catalytic Cys(41) by Sec, as established by mass spectrometry, while preserving the functional integrity of the peroxidase. The recombinant enzyme, whose synthesis is selenium-dependent, displayed a 4-fold enhanced peroxidase activity as compared with the Cys-containing analog, thus confirming the higher catalytic power of Sec compared with Cys in cit-PHGPx active site. The study led also to refinement of the minimal sequence requirements of the bacterial-type SECIS, and, for the first time, to the heterologous expression in Escherichia coli of a eukaryotic selenoprotein containing a SECIS in its open reading frame.
Hazebrouck
S
S
Department of Fruit Tree Breeding and Molecular Genetics, Agricultural Research Organization, The Volcani Center, 50250 Bet-Dagan, Israel.
Camoin
L
L
Faltin
Z
Z
Strosberg
A D
AD
Eshdat
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D002957:Citrus / Q000201:enzymology*; D003545:Cysteine; D004926:Escherichia coli / Q000235:genetics*; D005979:Glutathione Peroxidase / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D015202:Protein Engineering; D012345:RNA, Transfer, Amino Acid-Specific / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis*; D017279:Selenocysteine; D013329:Structure-Activity Relationship
D012345:RNA, Transfer, Amino Acid-Specific; D011994:Recombinant Proteins; C036928:tRNA, selenocysteine-; D017279:Selenocysteine; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D005979:Glutathione Peroxidase; D003545:Cysteine