Genes and biochemical characterization of three novel chlorophyllase isozymes from Brassica oleracea

Guan Chiun Lee, Hanna Chepyshko, Hsiu Hui Chen, Chih Chieh Chu, Yi Fan Chou, Casimir C. Akoh, Jei Fu Shaw

研究成果: 雜誌貢獻期刊論文同行評審

18 引文 斯高帕斯(Scopus)

摘要

Three full length cDNAs (BoCLH1, 1140 bp; BoCLH2, 1104 bp; BoCLH3, 884 bp) encoding putative chlorophyllases were cloned from the cDNA pools of broccoli (Brassica oleracea) florets and characterized. The amino acid sequence analysis indicated that these three BoCLHs contained a highly conserved lipase motif (GXSXG). However, only BoCLH3 lacked the His residue which is the component of the catalytic triad (Ser-His-Asp). N-terminal sequences of BoCLH1 and BoCLH2 were predicted to have typical signal sequences for the chloroplast, whereas the plasma membrane-targeting sequence was identified in BoCLH3. The predicted molecular masses of BoCLH1, 2, and 3 were 34.7, 35.3, and 23.5 kDa, respectively. The recombinant BoCLHs were successfully expressed in Escherichia coli for the biochemical characterization. The recombinant BoCLH3 showed very low chlorophyllase activity possibly due to its incomplete catalytic triad. BoCLH1 and BoCLH2 showed significant differences in biochemical properties such as pH stability and temperature optimum. Kinetic analysis revealed that BoCLH1 preferably hydrolyzed Mg-free chlorophyll, while BoCLH2 hydrolyzed both chlorophyll and Mg-free chlorophyll at a similar level. Different characteristics between BoCLH1 and BoCLH2 implied that they may have different physiological functions in broccoli. The catalytic triad of recombinant BoCLH2 was identified as Ser141, His247, and Asp170 by site-directed mutagenesis. It suggested that the three broccoli chlorophyllase isozymes were serine hydrolases.

原文英語
頁(從 - 到)8651-8657
頁數7
期刊Journal of Agricultural and Food Chemistry
58
發行號15
DOIs
出版狀態已發佈 - 2010 八月 11

ASJC Scopus subject areas

  • 化學 (全部)
  • 農業與生物科學 (全部)

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