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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 BrAOP2 heterologously expressed in E. coli. HPLC results (monitored at 229nm) of purified desulfoglucosinolates from bacterial extracts containing heterologously expressed BrAOP2 fusion proteins is shown. The compound identities were confirmed by comparing retention times and UV light absorption profiles with those of authentic standards. Std 2 indicates desulfated GRA standard, Std 1 indicates desulfated NAP standard, BrAOP2.1, BrAOP2.2, and BrAOP2.3 indicate GRA treated with these three BrAOP2 enzymes, and H2O shows GRA treated with ddH2O as the negative control.
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Figure 5: Enzymatic activity of BrAOP2 heterologously expressed in E. coli. HPLC results (monitored at 229nm) of purified desulfoglucosinolates from bacterial extracts containing heterologously expressed BrAOP2 fusion proteins is shown. The compound identities were confirmed by comparing retention times and UV light absorption profiles with those of authentic standards. Std 2 indicates desulfated GRA standard, Std 1 indicates desulfated NAP standard, BrAOP2.1, BrAOP2.2, and BrAOP2.3 indicate GRA treated with these three BrAOP2 enzymes, and H2O shows GRA treated with ddH2O as the negative control.

Mentions: The in vitro catalytic activity of the three BrAOP2 proteins can be monitored readily when they are expressed heterologously by measuring the conversion of GRA to 3-butenyl glucosinolate (NAP). We therefore heterologously expressed and purified thioredoxin fusion proteins for the three BrAOP2 genes in E. coli. As shown in Fig. 5, all three BrAOP2 proteins successfully catalysed the conversion of GRA to NAP, showing that they all had the capacity to convert methylsulfinylalkyl glucosinolates to the alkenyl form (GSL-ALKs).


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 BrAOP2 heterologously expressed in E. coli. HPLC results (monitored at 229nm) of purified desulfoglucosinolates from bacterial extracts containing heterologously expressed BrAOP2 fusion proteins is shown. The compound identities were confirmed by comparing retention times and UV light absorption profiles with those of authentic standards. Std 2 indicates desulfated GRA standard, Std 1 indicates desulfated NAP standard, BrAOP2.1, BrAOP2.2, and BrAOP2.3 indicate GRA treated with these three BrAOP2 enzymes, and H2O shows GRA treated with ddH2O as the negative control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: Enzymatic activity of BrAOP2 heterologously expressed in E. coli. HPLC results (monitored at 229nm) of purified desulfoglucosinolates from bacterial extracts containing heterologously expressed BrAOP2 fusion proteins is shown. The compound identities were confirmed by comparing retention times and UV light absorption profiles with those of authentic standards. Std 2 indicates desulfated GRA standard, Std 1 indicates desulfated NAP standard, BrAOP2.1, BrAOP2.2, and BrAOP2.3 indicate GRA treated with these three BrAOP2 enzymes, and H2O shows GRA treated with ddH2O as the negative control.
Mentions: The in vitro catalytic activity of the three BrAOP2 proteins can be monitored readily when they are expressed heterologously by measuring the conversion of GRA to 3-butenyl glucosinolate (NAP). We therefore heterologously expressed and purified thioredoxin fusion proteins for the three BrAOP2 genes in E. coli. As shown in Fig. 5, all three BrAOP2 proteins successfully catalysed the conversion of GRA to NAP, showing that they all had the capacity to convert methylsulfinylalkyl glucosinolates to the alkenyl form (GSL-ALKs).

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.