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Three genes encoding AOP2, a protein involved in aliphatic glucosinolate biosynthesis, are differentially expressed in Brassica rapa.

Zhang J, Liu Z, Liang J, Wu J, Cheng F, Wang X - J. Exp. Bot. (2015)

Bottom Line: Site-directed mutagenesis showed that His356, Asp310, and Arg376 residues are required for the catalytic activity of one of the BrAOP2 proteins (BrAOP2.1).Promoter-β-glucuronidase lines revealed that the BrAOP2.3 gene displayed an overlapping but distinct tissue- and cell-specific expression profile compared with that of the BrAOP2.1 and BrAOP2.2 genes.Taken together, our results revealed that all three BrAOP2 paralogues are active in B. rapa but have functionally diverged.

View Article: PubMed Central - PubMed

Affiliation: Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Zhongguancun Nandajie No. 12, Haidian District, Beijing 100081, PR China.

No MeSH data available.


Enzymatic activity of heterologously expressed site-directed mutant BrAOP2.1 proteins. The purified mutant proteins were assayed with GRA and the products were extracted and analysed by HPLC as described in Materials and methods. Site-directed mutagenesis of BrAOP2.1 was conducted four times to test the four active-site residues. (This figure is available in colour at JXB online.)
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Figure 8: Enzymatic activity of heterologously expressed site-directed mutant BrAOP2.1 proteins. The purified mutant proteins were assayed with GRA and the products were extracted and analysed by HPLC as described in Materials and methods. Site-directed mutagenesis of BrAOP2.1 was conducted four times to test the four active-site residues. (This figure is available in colour at JXB online.)

Mentions: To test the hypothesis that strictly conserved amino acid residues are required for AOP2 activity, we mutated the nucleotides that encode the conserved amino acids of BrAOP2.1 by site-directed mutagenesis. The Asp310 codon was changed to encode alanine (D310A), the histidine codons at positions 308 and 356 were changed to encode leucine (H308L and H356L), and the codons for arginine residue 376 were changed to encode tryptophan (R376W). Heterologous expression and enzyme assays were conducted for the mutant proteins, and the purified H308L mutant enzyme was shown to still catalyse the conversion of GRA to NAP, suggesting that His308 may be not essential for the catalytic mechanism (Fig. 8). By contrast, when His356, Asp310, or Arg376 were changed to leucine, alanine, and tryptophan, respectively, no catalytic activity was detected in any of the mutant BrAOP2.1 proteins (Fig. 8). These results suggested that these three highly conserved amino acid residues shown to be necessary for iron binding in the 2OG-FeII_Oxy domain are crucial for enzymatic activity in BrAOP2.


Three genes encoding AOP2, a protein involved in aliphatic glucosinolate biosynthesis, are differentially expressed in Brassica rapa.

Zhang J, Liu Z, Liang J, Wu J, Cheng F, Wang X - J. Exp. Bot. (2015)

Enzymatic activity of heterologously expressed site-directed mutant BrAOP2.1 proteins. The purified mutant proteins were assayed with GRA and the products were extracted and analysed by HPLC as described in Materials and methods. Site-directed mutagenesis of BrAOP2.1 was conducted four times to test the four active-site residues. (This figure is available in colour at JXB online.)
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4588880&req=5

Figure 8: Enzymatic activity of heterologously expressed site-directed mutant BrAOP2.1 proteins. The purified mutant proteins were assayed with GRA and the products were extracted and analysed by HPLC as described in Materials and methods. Site-directed mutagenesis of BrAOP2.1 was conducted four times to test the four active-site residues. (This figure is available in colour at JXB online.)
Mentions: To test the hypothesis that strictly conserved amino acid residues are required for AOP2 activity, we mutated the nucleotides that encode the conserved amino acids of BrAOP2.1 by site-directed mutagenesis. The Asp310 codon was changed to encode alanine (D310A), the histidine codons at positions 308 and 356 were changed to encode leucine (H308L and H356L), and the codons for arginine residue 376 were changed to encode tryptophan (R376W). Heterologous expression and enzyme assays were conducted for the mutant proteins, and the purified H308L mutant enzyme was shown to still catalyse the conversion of GRA to NAP, suggesting that His308 may be not essential for the catalytic mechanism (Fig. 8). By contrast, when His356, Asp310, or Arg376 were changed to leucine, alanine, and tryptophan, respectively, no catalytic activity was detected in any of the mutant BrAOP2.1 proteins (Fig. 8). These results suggested that these three highly conserved amino acid residues shown to be necessary for iron binding in the 2OG-FeII_Oxy domain are crucial for enzymatic activity in BrAOP2.

Bottom Line: Site-directed mutagenesis showed that His356, Asp310, and Arg376 residues are required for the catalytic activity of one of the BrAOP2 proteins (BrAOP2.1).Promoter-β-glucuronidase lines revealed that the BrAOP2.3 gene displayed an overlapping but distinct tissue- and cell-specific expression profile compared with that of the BrAOP2.1 and BrAOP2.2 genes.Taken together, our results revealed that all three BrAOP2 paralogues are active in B. rapa but have functionally diverged.

View Article: PubMed Central - PubMed

Affiliation: Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Zhongguancun Nandajie No. 12, Haidian District, Beijing 100081, PR China.

No MeSH data available.